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AU2018267693B2 - Biomarkers and uses thereof - Google Patents

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AU2018267693B2
AU2018267693B2 AU2018267693A AU2018267693A AU2018267693B2 AU 2018267693 B2 AU2018267693 B2 AU 2018267693B2 AU 2018267693 A AU2018267693 A AU 2018267693A AU 2018267693 A AU2018267693 A AU 2018267693A AU 2018267693 B2 AU2018267693 B2 AU 2018267693B2
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Flavia Huygens
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Microbio Pty Ltd
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Abstract

The present invention generally relates to methods and agents for identifying and/or classifying microbes (especially bacteria), yeast organisms and filamentous fungi. In particular, the present invention concerns the discovery of unique single nucleotide polymorphisms (SNPs) in bacterial 16S ribosomal RNA (16S rRNA) and yeast organism and filamentous fungi 18S ribosomal RNA, and methods of classifying and/or identifying bacteria, yeast organisms and/or filamentous fungi in a sample based on the presence of one or more of those SNPs. The present invention also concerns probes, primers and kits that are useful in those methods.

Description

BIOMARKERS AND USES THEREOF TECHNICAL FIELD
[0001] The present invention relates generally to methods and agents for identifying and/or classifying microbes. The methods and agents are based on the detection of polymorphisms within the 16S ribosomal RNA gene or gene products for bacteria, and within the 18S ribosomal RNA gene or gene products for yeast organisms and filamentous fungi. The invention also features methods for the treatment of infections of bacteria, yeast organisms or filamentous fungi based on the diagnostic methods of the present invention.
BACKGROUND
[0002] Rapid and accurate identification or classification of microbes (such as bacteria), yeast organisms and filamentous fungi in a sample is highly desirable.
[0003] First and foremost, rapid and accurate diagnosis of such infections can make the difference between life and death of a patient by allowing early implementation of an effective treatment or therapy.
[0004] Rapid and accurate identification or classification may assist in the implementation of effective control measures to manage, control, eradicate and/or eliminate microbes, yeast organisms or filamentous fungi in contaminated solutions, materials or foodstuffs, which may otherwise pose a threat to the wellbeing of organisms or the quality of production of the solutions, materials or foodstuffs.
[0005] Also, rapid and accurate identification or classification may assist with natural microbial, yeast organisms or filamentous fungi populations in a sample, such as, e.g., for ecological studies of microbial diversity, phylum spectrum and/or relative phylum abundance, or for monitoring deviation of a balance from a normal state in pathological conditions for an organism, such as, e.g., enteric, respiratory and skin disorders.
[0006] It may also be desirable to rapidly and accurately identify or classify microbes, yeast or filamentous fungi post therapy, treatment or modulation. For example, rapid microbial identification or classification may be of use in analysing the use of antibiotics, steroids, immune modulators, pre- and post-biotics, soil or water treatments, filtration, sterilization procedures and antiseptics.
[0007] Currently most diagnostic techniques commercially available typically require isolation and identification of live microbes, yeast organisms and filamentous fungi from a sample using culture, but this technique is negatively affected by considerable turn around time and suboptimal sensitivity, specificity and predictive value. It also can require more extensive handling of the organism, which can be particularly undesirable for some organisms such as security sensitive biological agents.
[0008] Alternative diagnostic techniques available include the use of polymerase chain reaction (PCR), particularly in conjunction with culture. However, a problem with such a technique is a lack of trust in early positive PCR results in the absence of culture results or relevant clinical or physical symptoms. Another problem with such a technique is, like with culture, a lack of sensitivity and specificity when attempting to detect small quantities of microbial nucleic acid in a background of host nucleic acid.
[0009] Hence, there is a recognized need for rapid and reliable techniques for accurate diagnosis of bacteria, yeast organisms and/or filamentous fungi.
SUMMARY OF INVENTION
[0010] In various aspects the present invention is predicated in part on high conservation of the 16S (Svedberg unit) ribosomal RNA (16S rRNA) gene between prokaryotes, including bacteria, and the high conservation of the 18S ribosomal RNA (18S rRNA) gene between yeast organisms and filamentous fungi, and also in part on the discovery of multiple single nucleotide polymorphisms (SNPs) within the 16S rRNA gene and 18S rRNA gene that may be useful in the identification and classification of microbes, particularly bacterial microbes, and yeast organisms and/or filamentous fungi in a sample.
[0011] Generally speaking, prokaryotes, including bacteria, contain 16S rRNA, which is a component of the 30S small subunit of the prokaryotic ribosome. The 16S rRNA is approximately 1,500 nucleotides in length and encoded by the 16S rRNA gene (also referred to as 16S rDNA), which is generally part of a co-transcribed operon also containing the 23S and 5S rRNA genes. Although the DNA sequence of the 16S rRNA genes (and thus the RNA sequence of the 16S rRNA molecules) is highly conserved between prokaryotes, there are regions of variation (Weisberg W.G., et al., 1991).
[0012] Similarly, the 18S rRNA gene in yeast and filamentous fungi is the homologue of the 16S rRNA gene in prokaryotes. The 18S rRNA is a component of the 40S small eukaryotic ribosomal subunit. The DNA sequence of the 18S rRNA gene (and thus the RNA sequence of the 18S rRNA molecules) is also highly conserved in yeast and filamentous fungi.
[0013] According to a first aspect of the invention, there is provided a method of identifying, partially identifying or classifying at least one bacterium, yeast organism or filamentous fungi in a sample. In one embodiment, said method comprises analysing at least a portion of a bacterial 16S rRNA gene or gene product from the sample, or at least a portion of a yeast organism or filamentous fungi 18S rRNA gene or gene product from the sample, for the presence or absence of at least one single nucleotide polymorphism (SNP), wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is at a position corresponding to at least one of positions 273, 378, 412, 440, 488, 647, and 653 of the 16S rRNA gene set forth in SEQ ID NO: 1; or wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is in or corresponds to the 16S rRNA gene set forth in SEQ ID NO: 43; or wherein the at least one bacterium, yeast organism or filamentous fungi in the sample is identified, partially identified or classified based on the presence or absence of the at least one SNP.
In another embodiment, said method comprises analysing at least a portion of a bacterial 16S rRNA gene or gene product from the sample, or at least a portion of a yeast organism or filamentous fungi 18S rRNA gene or gene product from the sample, for the presence or absence of at least one single nucleotide polymorphism (SNP), wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is at a position corresponding to at least one of positions 273, 378, 412, 440, 488, 647, 653, 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1; wherein the at least one bacterium, yeast organism or filamentous fungi in the sample is identified, partially identified or classified based on the presence or absence of the at least one SNP.
[0014] According to a second aspect of the present invention, there is provided a method of identifying, partially identifying, classifying or diagnosing a bacterial, yeast organism or filamentous fungi infection in a subject. In one embodiment, said method comprises analysing the presence or absence of at least one single nucleotide polymorphism (SNP) in at least a portion of a bacterial 16S rRNA gene or gene product or in at least a portion of a yeast organism or filamentous fungi 18S rRNA gene or gene product in a sample from the subject; wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is at a position corresponding to at least one of positions 273, 378, 412, 440, 488,
647, 653 of the 16S rRNA gene set forth in SEQ ID NO: 1; or wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is in or corresponds to the 16S rRNA gene set forth in SEQ ID NO: 43; or wherein the presence or absence of said at least one SNP in the at least a portion of the 16S rRNA gene or gene product or in the at least a portion of the 18S rRNA gene or gene product is used to identify, partially identify, classify or diagnose the bacterial, yeast organism or filamentous fungi infection in the subject.
In another embodiment, said method comprises analysing the presence or absence of at least one single nucleotide polymorphism (SNP) in at least a portion of a bacterial 16S rRNA gene or gene product or in at least a portion of a yeast organism or filamentous fungi 18S rRNA gene or gene product in a sample from the subject; wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is at a position corresponding to at least one of positions 273, 378, 412, 440, 488, 647, 653, 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1; wherein the presence or absence of said at least one SNP in the at least a portion of the 16S rRNA gene or gene product or in the at least a portion of the 18S rRNA gene or gene product is used to identify, partially identify, classify or diagnose the bacterial, yeast organism or filamentous fungi infection in the subject.
[0015] According to a third aspect of the present invention, there is provided method of treating a subject having a bacterial, yeast organism or filamentous fungi infection, said method comprising: identifying, partially identifying, classifying or diagnosing a bacterial, yeast organism or filamentous fungi infection in a subject according to the method of the second aspect, to identify, partially identify, classify or diagnose the bacterial, yeast organism or filamentous fungi infection in the subject; administering to the subject a therapy or treatment agent for treating the bacterial, yeast organism or filamentous fungi infection in the subject.
[0016] According to a fourth aspect of the present invention, there is provided at least one isolated probe, tool or reagent.
[0017] In one embodiment of the fourth aspect, said at least one isolated probe, tool or reagent is capable of identifying, partially identifying, or classifying at least one bacteria, yeast organism or filamentous fungi in a sample, wherein the probe, tool or reagent is capable of binding, detecting or identifying the presence or absence of at least one single nucleotide polymorphism (SNP) in at least a portion of a bacterial 16S rRNA gene or gene product or at least a portion of a yeast organism or filamentous fungi 18S rRNA gene or gene product, wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is at a position corresponding to at least one of positions 273, 378, 412, 440, 488, 647, 653 of the 16S rRNA gene set forth in SEQ ID NO: 1; or wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is in or corresponds to the 16S rRNA gene set forth in SEQ ID NO: 43.
[0018] In another embodiment of the fourth aspect, said at least one isolated probe, tool or reagent is capable of identifying, partially identifying, or classifying at least one bacteria, yeast organism or filamentous fungi in a sample, wherein the probe, tool or reagent is capable of binding, detecting or identifying the presence or absence of at least one single nucleotide polymorphism (SNP) in at least a portion of a bacterial 16S rRNA gene or gene product or at least a portion of a yeast organism or filamentous fungi 18S rRNA gene or gene product, wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is at a position corresponding to at least one of positions 273, 378, 412, 440, 488, 647, 653, 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1.
[0019] According to another embodiment of the fourth aspect, said at least one isolated probe, tool or reagent is capable of discriminating between a sample that comprises at least one bacteria, yeast organism or filamentous fungi and a sample that does not comprise at least one bacteria, yeast organism or filamentous fungi, wherein the probe, tool or reagent is capable of binding, detecting or identifying the presence or absence of at least one single nucleotide polymorphism (SNP) in at least a portion of a bacterial 16S rRNA gene or gene product or at least a portion of a yeast organism or filamentous fungi 18S rRNA gene or gene product, wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is at a position corresponding to at least one of positions 273, 378, 412, 440, 488, 647, 653 of the 16S rRNA gene set forth in SEQ ID NO: 1; or wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is in or corresponds to the 16S rRNA gene set forth in SEQ ID NO: 43.
[0020] According to another embodiment of the fourth aspect, said at least one isolated probe, tool or reagent is capable of discriminating between a sample that comprises at least one bacteria, yeast organism or filamentous fungi and a sample that does not comprise at least one bacteria, yeast organism or filamentous fungi, wherein the probe, tool or reagent is capable of binding, detecting or identifying the presence or absence of at least one single nucleotide polymorphism (SNP) in at least a portion of a bacterial 16S rRNA gene or gene product or at least a portion of a yeast organism or filamentous fungi 18S rRNA gene or gene product, wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is at a position corresponding to at least one of positions 273, 378, 412, 440, 488, 647, 653, 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1.
[0021] According to a fifth aspect of the present invention, there is provided a method of identifying, partially identifying, or classifying at least one bacterium, yeast organism or filamentous fungi in a sample. In one embodiment, said method comprises: combining with the sample the at least one isolated probe, tool or reagent of the fourth aspect; and identifying, partially identifying, or classifying the at least one bacterium, yeast organism or filamentous fungi based on the presence or absence of at least one at least one single nucleotide polymorphism (SNP) in at least a portion of a bacterial 16S rRNA gene or gene product or at least a portion of a yeast organism or filamentous fungi 18S rRNA gene or gene product, wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is at a position corresponding to at least one of positions 273, 378, 412, 440, 488, 647, 653 of the 16S rRNA gene set forth in SEQ ID NO: 1; or wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is in or corresponds to the 16S rRNA gene set forth in SEQ ID NO: 43.
In another embodiment, said method comprises: combining with the sample the at least one isolated probe, tool or reagent of the fourth aspect; and identifying, partially identifying, or classifying the at least one bacterium, yeast organism or filamentous fungi based on the presence or absence of at least one at least one single nucleotide polymorphism (SNP) in at least a portion of a bacterial 16S rRNA gene or gene product or at least a portion of a yeast organism or filamentous fungi 18S rRNA gene or gene product, wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is at a position corresponding to at least one of positions 273, 378, 412, 440, 488, 647, 653, 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1.
[0022] According to a sixth aspect of the present invention, there is provided an array
(especially a microarray) comprising more than one said isolated probe, tool or reagent of the fourth aspect.
[0023] According to a seventh aspect of the present invention, there is provided a biochip comprising a solid substrate and at least one isolated probe, tool or reagent of the fourth aspect.
[0024] According to an eighth aspect of the present invention, there is provided a kit or assay.
[0025] In one embodiment of the eighth aspect, the kit or assay is for classifying or identifying at least one bacterium or at least one yeast organism or filamentous fungi in a sample, said kit or assay comprising: the at least one probe, tool or reagent of the fourth aspect; the array of the sixth aspect; and/or the biochip of the seventh aspect.
[0026] In another embodiment of the eighth aspect, the kit or assay is capable of discriminating between a sample that comprises at least one bacteria, yeast organism or filamentous fungi and a sample that does not comprise at least one bacteria, yeast organism or filamentous fungi, wherein the kit or assay comprises the probe, tool or reagent of the fourth aspect.
[0027] According to a ninth aspect of the present invention, there is provided at least one single nucleotide polymorphism (SNP). In one embodiment, said at least one SNP is in at least a portion of a bacterial 16S rRNA gene or gene product or at least a portion of a yeast organism or filamentous fungi 18S rRNA gene or gene product for use as an indicator for classifying, identifying or partially identifying at least one bacterium, yeast organism or filamentous fungi in a sample; wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is at a position corresponding to at least one of positions 273, 378, 412, 440, 488, 647, 653 of the 16S rRNA gene set forth in SEQ ID NO: 1; or wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is in or corresponds to the 16S rRNA gene set forth in SEQ ID NO: 43.
In another embodiment, said at least one SNP is in at least a portion of a bacterial 16S rRNA gene or gene product or at least a portion of a yeast organism or filamentous fungi 18S rRNA gene or gene product for use as an indicator for classifying, identifying or partially identifying at least one bacterium, yeast organism or filamentous fungi in a sample; wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is at a position corresponding to at least one of positions 273, 378, 412, 440, 488,
647, 653, 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1.
[0028] According to a tenth aspect of the present invention, there is provided a use of at least one single nucleotide polymorphism (SNP) in at least a portion of a bacterial 16S rRNA gene or gene product or at least a portion of a yeast organism or filamentous fungi 18S rRNA gene or gene product as defined in the first aspect, or the at least one isolated probe, tool or reagent of the fourth aspect in identifying, partially identifying or classifying at least one bacterium, yeast organism or filamentous fungi in a sample, wherein said at least one bacterium, yeast organism or filamentous fungi is identified, partially identified or classified based on the presence or absence of at least one SNP in at least a portion of the 16S rRNA gene or gene product or of at least a portion of the 18S rRNA gene or gene product from the sample.
[0029] According to an eleventh aspect of the present invention, there is provided a kit comprising a probe, tool or reagent for use in the method of any one of the first, second, third or fifth aspects of the present invention.
[0030] Further aspects of the invention are provided below.
[0031] According to a twelfth aspect of the present invention, there is provided at least one single nucleotide polymorphism (SNP) in a 16S rRNA gene for use or when used in identifying at least one bacterium in a sample or classifying bacteria in the sample; or at least one single nucleotide polymorphism (SNP) in a 18S rRNA gene for use or when used in identifying at least one yeast organism or filamentous fungi in a sample or classifying a yeast organism or filamentous fungi in the sample.
[0032] According to a thirteenth aspect of the present invention, there is provided at least one probe, tool or reagent for use or when used in identifying at least one bacterium in a sample or classifying bacteria in the sample, said at least one probe, tool or reagent is capable of specifically binding, detecting or identifying at least a portion of a 16S rRNA gene in the sample containing at least one SNP; or at least one probe, tool or reagent for use or when used in identifying at least one yeast organism or filamentous fungi in a sample or classifying at least one yeast organism or filamentous fungi in the sample, said at least one probe, tool or reagent is capable of specifically binding, detecting or identifying at least a portion of a 18S rRNA gene in the sample containing at least one SNP.
[0033] According to a fourteenth aspect of the present invention, there is provided at least one probe, tool or reagent for use or when used in identifying a bacterium in a sample or classifying bacteria in the sample, said at least one probe, tool or reagent comprising an oligonucleotide having a nucleotide sequence as set forth in any one of the of SEQ ID NOs:16 35, 56 or 57; or at least one probe, tool or reagent for use or when used in identifying at least one yeast organism or filamentous fungi in a sample or classifying at least one yeast organism or filamentous fungi in the sample, said at least one probe, tool or reagent comprising an oligonucleotide having a nucleotide sequence as set forth in any one of the of SEQ ID NOs:53 55.
[0034] According to a fifteenth aspect of the present invention, there is provided a use of the at least one SNP as defined in the twelfth aspect or the at least one probe, tool or reagent as defined in the thirteenth or fourteenth aspect in identifying at least one bacterium in a sample or classifying bacteria in the sample, wherein said at least one bacterium is identified or bacteria classified based on the presence of at least one SNP in a 16S rRNA gene from the sample as described above; or a use of the at least one SNP as defined in the twelfth aspect or the at least one probe, tool or reagent as defined in the thirteenth or fourteenth aspect in identifying at least one yeast organism or filamentous fungi in a sample or classifying at least one yeast organism or filamentous fungi in the sample, wherein said at least one yeast organism or filamentous fungi is identified or classified based on the presence of at least one SNP in a 18S rRNA gene from the sample as described above.
[0035] According to a sixteenth aspect of the present invention, there is provided a method of identifying at least one bacterium in a sample, said method comprising analysing a 16S rRNA gene from the sample for the at least one SNP as defined in the first aspect, wherein the at least one bacterium is identified based on the presence of the at least one SNP; or a method of identifying at least one yeast organism or filamentous fungi in a sample, said method comprising analysing a 18S rRNA gene from the sample for the at least one SNP as defined in the twelfth aspect, wherein the at least one yeast organism or filamentous fungi is identified based on the presence of the at least one SNP.
[0036] In one embodiment there is provided a method of identifying at least one bacterium in a sample, said method comprising analysing nucleic acid from the sample for at least one SNP in a bacterial 16S rRNA gene at a position corresponding to at least one of positions 273, 378, 412, 440, 488, 647, and 653 of the 16S rRNA gene set forth in SEQ ID NO: 1, wherein the at least one bacterium is identified based on the presence of the at least one SNP.
[0037] According to a seventeeth aspect of the present invention, there is provided a method of classifying bacteria in a sample, said method comprising analysing a 16S rRNA gene from the sample for the at least one SNP as defined in the first aspect, wherein the bacteria are classified based on the presence of the at least one SNP; or a method of classifying at least one yeast organism or filamentous fungi in a sample, said method comprising analysing a 18S rRNA gene from the sample for the at least one SNP as defined in the twelfth aspect, wherein the at least one yeast organism or filamentous fungi are classified based on the presence of the at least one SNP.
[0038] According to an eighteenth aspect of the present invention, there is provided a method of identifying at least one bacterium in a sample, said method comprising: combining with the sample the at least one probe, tool or reagent as defined in the thirteenth or fourteenth aspects; and identifying the at least one bacterium based on the presence of at least one SNP in a 16S rRNA gene from the sample as broadly described above; or
a method of identifying at least one yeast organism or filamentous fungi in a sample, said method comprising: combining with the sample the at least one probe, tool or reagent as defined in the thirteenth or fourteenth aspects; and identifying the at least one yeast organism or filamentous fungi based on the presence of at least one SNP in a 18S rRNA gene from the sample as broadly described above.
[0039] According to a nineteenth aspect of the present invention, there is provided a method of classifying bacteria in a sample, said method comprising: combining with the sample the at least one probe, tool or reagent as defined in the thirteenth or fourteenth aspects; and classifying the bacteria based on the presence of at least one SNP in a 16S rRNA gene from the sample as broadly described above; or
a method of classifying at least one yeast organism or filamentous fungi in a sample, said method comprising: combining with the sample the at least one probe, tool or reagent as defined in the thirteenth or fourteenth aspects; and classifying the at least one yeast organism or filamentous fungi based on the presence of at least one SNP in a 18S rRNA gene from the sample as broadly described above.
[0040] According to a twentieth aspect of the present invention, there is provided a method of diagnosing a bacterial infection in a subject, said method comprising analysing a 16S rRNA gene from a sample obtained from the subject for the at least one SNP as defined in the twelfth aspect, wherein the bacterial infection is diagnosed by identifying at least one causative bacterium in the sample based on the presence of the at least one SNP; or a method of diagnosing at least one yeast organism or filamentous fungi infection in a subject, said method comprising analysing a 18S rRNA gene from a sample obtained from the subject for the at least one SNP as defined in the twelfth aspect, wherein the at least one yeast organism or filamentous fungi infection is diagnosed by identifying at least one causative yeast organism or filamentous fungi in the sample based on the presence of the at least one SNP.
[0041] According to a twenty-first aspect of the present invention, there is provided a method of diagnosing a bacterial infection in a subject, said method comprising: combining with a sample obtained from the subject the at least one probe, tool or reagent as defined in the thirteenth or fourteenth aspects; and diagnosing the bacterial infection by identifying at least one causative bacterium in the sample based on the presence of the at least one SNP in a 16S rRNA gene from the sample as broadly described above; or
a method of diagnosing at least one yeast organism or filamentous fungi infection in a subject, said method comprising: combining with a sample obtained from the subject the at least one probe, tool or reagent as defined in the thirteenth or fourteenth aspects; and diagnosing the at least one yeast organism or filamentous fungi infection by identifying at least one causative bacterium in the sample based on the presence of the at least one SNP in a 18S rRNA gene from the sample as broadly described above.
[0042] According to a twenty-second aspect of the present invention, there is provided a method of treating a subject having a bacterial infection, said method comprising: diagnosing the bacterial infection by identifying the at least one causative bacterium in the sample according to the method as defined in the twentieth or twenty-first aspects; and administering to the subject a therapy or treatment agent for treating the at least one causative bacterium identified to thereby treat the bacterial infection; or
a method of treating a subject having at least one yeast organism or filamentous fungi infection, said method comprising: diagnosing the at least one yeast organism or filamentous fungi infection by identifying the at least one causative bacterium in the sample according to the method as defined in the twentieth or twenty-first aspects; and administering to the subject a therapy or treatment agent for treating the at least one causative yeast organism or filamentous fungi identified to thereby treat the bacterial infection.
[0043] According to a twenty-third aspect of the present invention, there is provided an array of oligonucleotide probes for identifying at least one bacterium in a sample or classifying bacteria in the sample, said probes comprising oligonucleotides which hybridize to at least one SNP in a 16S rRNA gene in the sample as broadly described above; or an array of oligonucleotide probes for identifying at least one yeast organism or filamentous fungi in a sample or classifying at least one yeast organism or filamentous fungi in the sample, said probes comprising oligonucleotides which hybridize to at least one SNP in a 18S rRNA gene in the sample as broadly described above.
[0044] According to a twenty-fourth aspect of the present invention, there is provided a microarray comprising oligonucleotide probes for identifying at least one bacterium in a sample or classifying bacteria in the sample, said probes comprising oligonucleotides which hybridize to at least one SNP in a 16S rRNA gene in the sample as broadly described above; or a microarray comprising oligonucleotide probes for identifying at least one yeast organism or filamentous fungi in a sample or classifying at least one yeast organism or filamentous fungi in the sample, said probes comprising oligonucleotides which hybridize to at least one SNP in a 18S rRNA gene in the sample as broadly described above.
[0045] According to a twenty-fifth aspect of the present invention, there is provided a biochip comprising a solid substrate and at least one oligonucleotide probe for identifying at least one bacterium in a sample or classifying bacteria in the sample, said at least one probe comprising an oligonucleotide which hybridize to at least one SNP in a 16S rRNA gene in the sample as broadly described above; or a biochip comprising a solid substrate and at least one oligonucleotide probe for identifying at least one yeast organism or filamentous fungi in a sample or classifying at least one yeast organism or filamentous fungi in the sample, said at least one probe comprising an oligonucleotide which hybridize to at least one SNP in a 18S rRNA gene in the sample as broadly described above.
[0046] According to a twenty-sixth aspect of the present invention, there is provided a kit or assay for identifying at least one bacterium in a sample or classifying bacteria in the sample, said kit or assay comprising: the at least one probe, tool or reagent as described above; the array of oligonucleotide probes as described above; the microarray as described above; and/or the biochip as described above; or a kit or assay for identifying at least one yeast organism or filamentous fungi in a sample or classifying at least one yeast organism or filamentous fungi in the sample, said kit or assay comprising: the at least one probe, tool or reagent as described above; the array of oligonucleotide probes as described above; the microarray as described above; and/or the biochip as described above.
[0047] According to a twenty-seventh aspect of the present invention, there is provided a method of identifying, partially identifying, classifying or diagnosing an infection in a subject. In one embodiment of the twenty-seventh aspect, said infection is a bacterial, yeast organism or filamentous fungi infection, said method comprising assaying a biological sample obtained from the subject for a property of at least one bacterial 16S rRNA gene or gene product or a portion thereof or at least one yeast organism or filamentous fungi 18S rRNA gene or gene product or a portion thereof, wherein said assay comprises detecting at least one single nucleotide polymorphism (SNP) in the at least one bacterial 16S rRNA gene or gene product or a portion thereof or at least one yeast organism or filamentous fungi 18S rRNA gene or gene product or a portion thereof, wherein the at least one SNP in the bacterial 16S rRNA gene or gene product or a portion thereof is at a position corresponding to at least one of positions 273, 378, 412, 440, 488, 647, and 653 of the 16S rRNA gene set forth in SEQ ID NO: 1; or wherein the at least one SNP in the bacterial 16S rRNA gene or gene product or a portion thereof is in or corresponds to the 16S rRNA gene set forth in SEQ ID NO: 43; or wherein the at least one bacteria, yeast organism or filamentous fungi in the sample is identified, partially identified, classified or diagnosed based on the presence or absence of the at least one SNP.
In another embodiment of the twenty-seventh aspect, said infection is a bacterial, yeast organism or filamentous fungi infection, said method comprising assaying a biological sample obtained from the subject for a property of at least one bacterial 16S rRNA gene or gene product or a portion thereof or at least one yeast organism or filamentous fungi 18S rRNA gene or gene product or a portion thereof, wherein said assay comprises detecting at least one single nucleotide polymorphism (SNP) in the at least one bacterial 16S rRNA gene or gene product or a portion thereof or at least one yeast organism or filamentous fungi 18S rRNA gene or gene product or a portion thereof, wherein the at least one SNP in the bacterial 16S rRNA gene or gene product or a portion thereof is at a position corresponding to at least one of positions 273, 378, 412, 440, 488, 647, 653, 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1; wherein the at least one bacteria, yeast organism or filamentous fungi in the sample is identified, partially identified, classified or diagnosed based on the presence or absence of the at least one SNP.
[0047a] According to a twenty-eighth aspect of the present invention, there is provided a method of identifying, partially identifying or classifying at least one bacterium in a sample, said method comprising analysing at least a portion of a bacterial 16S rRNA gene or gene product from the sample for the presence or absence of at least two single nucleotide polymorphisms (SNPs);
wherein the at least two SNPs in the at least a portion of the bacterial 16S rRNA gene or gene product are at positions corresponding to at least two of positions 273, 378, 412, 440, 488, 647, 653, 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1; and
wherein the at least one bacterium in the sample is identified, partially identified or classified based on the presence or absence of the at least two SNPs.
[0047b] According to a twenty-ninth aspect of the present invention, there is provided a method of identifying, partially identifying, classifying or diagnosing a bacterial infection in a subject, said method comprising analysing for the presence or absence of at least two single nucleotide polymorphisms (SNPs) in at least a portion of a bacterial 16S rRNA gene or gene product in a sample from the subject;
wherein the at least two SNPs in the at least a portion of the bacterial 16S rRNA gene or gene product are at positions corresponding to at least two of positions 273, 378, 412, 440, 488, 647, 653, 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1; and
wherein the presence or absence of said at least two SNPs in the at least a portion of the 16S rRNA gene or gene product is used to identify, partially identify, classify or diagnose the bacterial infection in the subject.
[0047c] According to a thirtieth aspect of the present invention, there is provided a method of treating a subject having a bacterial infection, said method comprising:
identifying, partially identifying, classifying or diagnosing a bacterial infection in a subject according to the method of the twenty-ninth aspect; and
administering to the subject a therapy or treatment agent for treating the bacterial infection in the subject.
14a
[0047d] According to a thirty-first aspect of the present invention, there is provided a method of identifying, partially identifying or classifying at least one bacterium in a sample, said method comprising analysing at least a portion of a bacterial 16S rRNA gene or gene product from the sample for the presence or absence of at least one single nucleotide polymorphism (SNP);
wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is at a position corresponding to at least one of positions 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1; and
wherein the at least one bacterium in the sample is identified, partially identified or classified based on the presence or absence of the at least one SNP.
[0047e] According to a thirty-second aspect of the present invention, there is provided a method of identifying, partially identifying, classifying or diagnosing a bacterial infection in a subject, said method comprising analysing for the presence or absence of at least one single nucleotide polymorphism (SNP) in at least a portion of a bacterial 16S rRNA gene or gene product in a sample from the subject;
wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is at a position corresponding to at least one of positions 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1; and
wherein the presence or absence of said at least one SNP in the at least a portion of the 16S rRNA gene or gene product is used to identify, partially identify, classify or diagnose the bacterial infection in the subject.
[0047f] According to a thirty-third aspect of the present invention, there is provided a method of identifying, partially identifying, or classifying at least one bacterium in a sample, said method comprising:
combining with the sample at least one oligonucleotide comprising a nucleotide sequence as set forth in at least one of SEQ ID NOs: 22, 25-28, 33-35, and 57, or at least one oligonucleotide consisting of a nucleotide sequence as set forth in at least one of SEQ ID NOs: 16-35, 56 and 57; and
14b
identifying, partially identifying, or classifying the at least one bacterium based on the presence or absence of at least two single nucleotide polymorphisms (SNPs) in at least a portion of a bacterial 16S rRNA gene or gene product,
wherein the at least two SNPs in the at least a portion of the bacterial 16S rRNA gene or gene product are at positions corresponding to at least two of positions 273, 378, 412, 440, 488, 647, 653, 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1.
[0047g] According to a thirty-fourth aspect of the present invention, there is provided an array comprising more than one oligonucleotide comprising a nucleotide sequence as set forth in at least one of SEQ ID NOs: 22, 25-28, 33-35, and 57, or more than one oligonucleotide consisting of a nucleotide sequence as set forth in at least one of SEQ ID NOs: 16-35, 56 and 57.
[0047h] According to a thirty-fifth aspect of the present invention, there is provided a biochip comprising a solid substrate and at least one oligonucleotide comprising a nucleotide sequence as set forth in at least one of SEQ ID NOs: 22, 25-28, 33-35, and 57, or at least one oligonucleotide consisting of a nucleotide sequence as set forth in at least one of SEQ ID NOs: 16-35, 56 and 57.
[0047i] According to a thirty-sixth aspect of the present invention, there is provided a kit or assay when used according to the method of any one of the twenty-eighth to thirty-third aspects, said kit or assay comprising: an oligonucleotide comprising a nucleotide sequence as set forth in at least one of SEQ ID NOs: 22, 25-28, 33-35, and 57, or an oligonucleotide consisting of a nucleotide sequence as set forth in at least one of SEQ ID NOs: 16-35, 56 and 57; the array of the thirty-fourth aspect; and/or the biochip of the thirty-fifth aspect.
[0048] Features of the first to twenty-seventh aspects of the present invention, where appropriate, may be as described below.
[0049] In one embodiment, said at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is selected from SNPs at positions corresponding to at least one of positions 273, 378, 412, 440, 488, 647, 653, 737, 755, 762 and 776 of the 16S rRNA gene as set forth in SEQ ID NO:1. In some embodiments, more than one SNP may be used in the methods of the present invention. For example, at least two SNPs, at least three SNPs, at least four
14c
SNPS, at least five SNPs, at least six SNPs, at least seven SNPs, at least eight SNPs, at least nine SNPs, at least ten SNPs, or even at least eleven SNPs may be used.
[0050] In one embodiment, said at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is selected from SNPs at positions corresponding to positions 273, 378, 412, 440, 488, 647 and 653 of the 16S rRNA gene as set forth in SEQ ID NO:1. In some embodiments, more than one SNP may be used in the methods of the present invention. For example, at least two SNPs, at least three SNPs, at least four SNPS, at least five SNPs, at least six SNPs, or even at least seven SNPs may be used.
[0051] In another embodiment, said at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product may be in or correspond to the 16S rRNA gene as set forth in SEQ ID NO: 43. The at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product set forth in SEQ ID NO: 43 may be at a position corresponding to at least one of positions 746, 764, 771, or 785 of the 16S rRNA gene set forth in SEQ ID NO: 43 (or positions 737, 755, 762, or 776 of the 16S rRNA gene as set forth in SEQ ID NO:1). At least one said SNP, at least two said SNPs, at least three said SNPs or at least four said SNPs may be used.
[0052] In another embodiment, the at least one SNP in the at least a portion of the yeast organism or filamentous fungi 18S rRNA gene or gene product may be in or correspond to the 18S rRNA gene set forth in SEQ ID NO: 37. The at least one SNP in the at least a portion of the yeast organism or filamentous fungi 18S rRNA gene or gene product may be at a position corresponding to at least one of positions 343, 371, 388, 416, and 467 of the 18S rRNA gene set forth in SEQ ID NO: 37. At least one said SNP, at least two said SNPs, at least three said SNPs, at least four said SNPs or at least five said SNPs may be used.
[0053] In a further embodiment, said method comprises analysing at least a portion of a
[Text continues on page 15] bacterial 16S rRNA gene or gene product from the sample, or at least a portion of a yeast organism or filamentous fungi 18S rRNA gene or gene product from the sample, for the presence or absence of: single nucleotide polymorphisms in the bacterial 16S rRNA gene at a position corresponding to positions 273, 378, 412, 440, 488, 647, and 653 of the 16S rRNA gene set forth in SEQ ID NO: 1; or single nucleotide polymorphisms in the bacterial 16S rRNA gene a position corresponding to positions 746, 764, 771, and 785 of the 16S rRNA gene set forth in SEQ ID NO: 43; or single nucleotide polymorphisms in the yeast organism or filamentous fungi 18S rRNA gene at a position corresponding to positions 343, 371, 388, 416, and 467 of the 18S rRNA gene set forth in SEQ ID NO: 37.
[0054] In a further embodiment, said method comprises analysing at least a portion of a bacterial 16S rRNA gene or gene product from the sample, or at least a portion of a yeast organism or filamentous fungi 18S rRNA gene or gene product from the sample, for the presence or absence of: single nucleotide polymorphisms in the at least a portion of the bacterial 16S rRNA gene or gene product at a position corresponding to at least four of positions 273, 378, 412, 440, 488, 647, 653, 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1; or single nucleotide polymorphisms in the yeast organism or filamentous fungi 18S rRNA gene at a position corresponding to positions 343, 371, 388, 416, and 467 of the 18S rRNA gene set forth in SEQ ID NO: 37.
[0055] In some embodiments, the bacterium or bacteria is or are selected from among mammalian (e.g., human) associated bacteria, soil associated bacteria and water associated bacteria. In particular embodiments, the bacterium or bacteria may be a sepsis-associated bacterium or bacteria.
[0056] In some particular embodiments, the bacterium or bacteria is or are selected from among at least one of: Acinetobacter spp.; Actinobaccillus spp.; Actinomadura spp.; Actinomyces spp.; Actinoplanes spp.; Aeromonas spp.; Agrobacterium spp.; Alistipes spp.; Anaerococcus spp.; Arthrobacterspp.; Bacillus spp.; Bacteroides spp.; Brucella spp.; Bulleidia spp.; Burkholderia spp.; Cardiobacterium spp.; Citrobacter spp.; Clostridium spp.; Cornyebacterium spp.; Dermatophilus spp.; Dorea spp; Enterobacter spp.; Enterococcus spp.; Erysipelothrix spp.; Escherichia spp.; Eubacterium spp.; Ewardsiella spp.; Faecalibacterium spp.; Filifactor spp.; Finegoldia spp.; Flavobacterium spp.; Francisella spp.; Gallicola spp.; Haemophilus spp.; Helococcus spp.; Holdemania spp.; Hyphomicrobium spp.; Klebsiella spp.; Lactobacillus spp.; Legionella spp.; Listeria spp.; Methylobacterium spp.; Micrococcus spp.; Micromonospora spp.; Mobiluncus spp.; Moraxella spp.; Morganella spp.; Mycobacterium spp.; Neisseria spp.; Nocardia spp.; Paenibacillusspp.; Parabacteroidesspp.; Pasteurellaspp.; Peptoniphilus spp.; Peptostreptococcus spp.; Planococcus spp.; Planomicrobium spp.; Plesiomonas spp.; Porphyromonas spp.; Prevotella spp.; Propionibacteriumspp.; Proteus spp.; Providentia spp.; Pseudomonas spp.; Ralstonia spp.; Rhodococcus spp.; Roseburia spp.; Ruminococcus spp.; Salmonella spp.; Sedimentibacter spp.; Serratia spp.; Shigella spp.; Solobacterium spp.; Sphingomonas spp.; Staphylococcus spp.; Stenotrophomonas spp.; Streptococcus spp.; Streptomyces spp.; Tissierella spp.; Vibrio spp.; and Yersinia spp.
[0057] In some more particular embodiments, the bacterium or bacteria is or are selected from among at least one of: Acinetobacter baumannii; Acinetobacter calcoaceticus; Bacteroides fragilis; Bacteroides vulgatus; Citrobacter freundii; Enterobacter aerogenes; Enterobacter cloacae; Enterococcus avium; Enterococcusfaecalis; Enterococcusfaecium; Escherichia coli; Klebsiella oxytoca; Klebsiella pneumoniae; Proteus mirabilis; Pseudomonas aeruginosa; Serratia marcescens; Staphylococcus aureus; Staphylococcus epidermidis; Staphylococcus hominis; Staphylococcus saprophyticus; Stenotrophomonas maltophilia; Streptococcus agalactiae; Streptococcus anginosus; Streptococcus constellatus; Streptococcus intermedius; Streptococcus milleri; Streptococcus mitis; Streptococcus mutans; Streptococcus oralis; Streptococcus pneumoniae; Streptococcus pyogenes; Streptococcus sanguinis; and Streptococcus sobrinus.
[0058] In particular embodiments, the bacterium or bacteria is or are selected from among at least one of: Acinetobacter calcoaceticus; Enterobacter aerogenes; Enterobacter cloacae; Enterococcus faecalis; Enterococcus faecium; Escherichia coli; Klebsiella pneumoniae; Proteus mirabilis; Pseudomonas aeruginosa; Serratia marcescens; Staphylococcus aureus; Staphylococcus epidermidis; Streptococcus agalactiae; Streptococcus pneumoniae; and Streptococcus pyogenes.
[0059] In particular embodiments, the bacterium or bacteria is or are selected from among at least one of: Acinetobacter calcoaceticus; Enterobacter aerogenes; Enterobacter cloacae; Enterococcus faecalis; Enterococcus faecium; Escherichia coli; Klebsiella pneumoniae; Proteus mirabilis; Pseudomonas aeruginosa; Serratia marcescens; Staphylococcus aureus; Staphylococcus epidermidis; Streptococcus agalactiae; Streptococcus pneumoniae;
Streptococcus pyogenes; Listeria monocytogenes; Clostridium perfringens; Corynebacterium jeikeium; Bacteroidesfragilis;Neisseria meningitides; Haemophilus influenzae; Salmonella sp.; and Staphylococcus epidermidis. In another embodiment, the bacterium or bacteria is or are selected from among at least one of: Acinetobacter calcoaceticus; Enterobacter aerogenes; Enterobacter cloacae; Enterococcus faecalis; Enterococcus faecium; Escherichia coli; Klebsiella pneumoniae; Proteus mirabilis; Pseudomonas aeruginosa; Serratia marcescens; Staphylococcus aureus; Staphylococcus epidermidis; Streptococcus agalactiae;Streptococcus pneumoniae; Streptococcus pyogenes; Listeria monocytogenes; Clostridium perfringens; Corynebacterium jeikeium; Bacteroides fragilis; Neisseria meningitides; Haemophilus influenzae; Salmonella sp.; Staphylococcus epidermidis; Bacillus anthracis, Clostridium botulinum, Yersinia pestis, Francisella tularensis, Vibrio cholerae, and Burkholderia pseudomallei.
[0060] In one embodiment the bacterium or bacteria is a Security Sensitive Biological Agent (SSBA). The SSBA may be a Tier 1 agent or a Tier 2 agent. Exemplary Tier 1 agents include one or more of:, Bacillus anthracis (Anthrax), and Yesinia pestis (Plague). Exemplary Tier 2 agents include one or more of: Clostridium botulinum (botulism, especially toxin producing strains); Francisellatularensis (Tularaemia);Salmonella Typhi (typhoid), and Vibrio cholerae (especially Cholera serotypes 01 or 0139). In one embodiment the bacterium or bacteria is or are selected from among at least one of the group consisting of: Bacillus anthracis, Clostridium botulinum, Yersinia pestis, Francisellatularensis, Vibrio cholerae, and Burkholderiapseudomallei.
[0061] In one embodiment the yeast organism may be selected from among at least one of: Candida spp., Cryptococcus spp and Rhodotorula spp. Exemplary Candida spp. may include at least one of Candida albicans, Candida tropicalis, Candida stellatoidea, Candida krusei, Candida parapsilosis, Candida glabrata, Candida guilliermondii, Candida viswanathii, Candida auris and Candida lusitaniae. Exemplary Cryptococcus spp. may include at least one of Cryptococcus neoformans and Cryptococcus gattii. An exemplary Rhodotorula spp. may be Rhodotorula mucilaginosa.
[0062] In one embodiment the filamentous fungi may be selected from at least one of: Aspergillus spp. and Fusarium spp. Exemplary Fusarium spp. may include at least one of Fusarium solani, Fusarium oxysporum, Fusarium verticillioides, Fusarium proliferatum, Fusarium avenaceum, Fusarium bubigeum, Fusarium culmorum, Fusarium graminearum, Fusarium langsethiae, Fusarium poae, Fusarium sporotrichioides, Fusarium tricinctum and
Fusarium virguliforme; especially at least one of Fusarium solani, Fusarium oxysporum, Fusariumverticillioides and Fusariumproliferatum. Exemplary Aspergillus spp. may include at least one of Aspergillus fumigatus, Aspergillus flavus, Aspergillus clavatus, Aspergillus lentulus, Aspergillus terreus, and Aspergillus nidulans; especially at least one of Aspergillus fumigatus, Aspergillusflavus, and Aspergillus clavatus; more especially Aspergillusfumigatus.
[0063] In one embodiment, the yeast organism or filamentous fungi is at least one of the group consisting of: Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata,Fusariumsp., Aspergillusfumigatus, and Cryptococcus neoformans.
[0064] In one embodiment, the bacteria, yeast organism or filamentous fungi is a human pathogen.
[0065] In some embodiments, the methods of the present invention may be used to analyse blood from a subject with systemic inflammatory response syndrome (SIRS) to determine the origin of the SIRS (for example bacteria, yeast organism or filamentous fungi). In other embodiments, the methods of the present invention may be used to determine whether a subject has sepsis having a bacterial, yeast organism or filamentous fungi infectious origin. In both embodiments, the methods of the present invention may be used to determine the presence of, differentiate and/or identify bacteria, yeast organism or filamentous fungi present in the sample.
[0066] SIRS is an overwhelming whole body reaction that may have an infectious aetiology or non-infectious aetiology (i.e., infection-negative SIRS, or inSIRS). Sepsis is SIRS that occurs during infection. Sepsis in this instance is diagnosed by a clinician (when there is suspected infection) or through culture of an organism. Both SIRS and sepsis are defined by a number of non-specific host response parameters including changes in heart and respiratory rate, body temperature and white cell counts (Levy et al., 2003; Reinhart et al., 2012).
[0067] In some embodiments, the at least one SNP or at least one probe, tool or reagent may be used to classify bacteria in a sample as Gram-positive bacterium or bacteria or Gram negative bacterium or bacteria.
[0068] For example, in some embodiments, the bacterium or bacteria may be classified as Gram-positive based on any one of the above SNPs, especially at at least one of positions 273, 378, 412, 440, 488, 647, and 653 of the 16S rRNA gene set forth in SEQ ID NO: 1. In one such embodiment, the bactreria or bacterium may be classified based on SNPs at positions corresponding to positions 273 and 653 of the 16S rRNA gene as set forth in SEQ ID NO:1, wherein the bacterium or bacteria is determined to be Gram-positive when there is an A at position 273 and a T at position 653.
[0069] For example, in another embodiment, the bacterium or bacteria may be classified as Gram-positive based on at least one SNP at a position corresponding to position 440 of the 16S rRNA gene as set forth in SEQ ID NO:1, wherein the bacterium or bacteria is determined to be Gram-positive when there is a T at position 440. Conversely, wherein the bacterium or bacteria is determined to be Gram-negative when there is not a T at position 440.
[0070] In yet other embodiments, the at least one SNP or at least one probe, tool or reagent may be used to classify groups of bacteria, yeast organisms or filamentous fungi in a sample.
[0071] For example, in some embodiments, the bacteria may be classified as belonging to a particular genus based on at least one SNP selected from the above SNPs. In one such embodiment, the bacteria may be classified as belonging to a particular genus based on at least one SNP selected from SNPs at positions corresponding to positions 412 and 647 of the 16S rRNA gene as set forth in SEQ ID NO:1. For example, the bacterium or bacteria in a sample may be classified as belonging to the Staphylococcus genus when there is a T at position 412. For example, the bacterium or bacteria in a sample may be classified as belonging to the Enterococcus genus when there is a G at position 647.
[0072] In yet other embodiments, the at least one SNP or at least one probe, tool or reagent may be used to identify a bacterium in a sample as described above.
[0073] For example, the bacterium Enterobacter cloacae may be identified in a sample based on at least one SNP at a position corresponding to position 653 of the 16S rRNA gene as set forth in SEQ ID NO:1, wherein the bacterium Enterobactercloacae is identified when there is a G at position 653.
[0074] For example, bacterium selected from Streptococcus pneumoniae, Streptococcus agalactiae and Streptococcus pyogenes may be identified in a sample based on SNPs at positions corresponding to positions 378 and 488 of the 16S rRNA gene as set forth in SEQ ID NO:1, wherein the bacterium is: Streptococcus pneumoniae when there is an A at position 378 and a T at position 488; Streptococcus agalactiaewhen there is an A at position 378 and an A 488; and Streptococcuspyogenes when there is a G at position 378 and an A at position 488.
[0075] For example, in one embodiment, bacterium selected from among Acinetobacter calcoaceticus; Enterobacter cloacae; Escherichia coli; Klebsiella pneumoniae; Proteus mirabilis;Pseudomonas aeruginosa;Streptococcus agalactiae;Streptococcus pneumoniae; and Streptococcus pyogenes may be identified in a sample based on SNPs at positions corresponding to positions 273, 378, 412, 440, 488, 647 and 653 of the 16S rRNA gene as set forth in SEQ ID NO:1, wherein the bacterium is: Acinetobacter calcoaceticus when there is an A at positions 273, 440 and 647; Enterobacter cloacae when there is a G at position 653; Escherichia coli when there is a T at position 273 and a T at position 653; Klebsiella pneumoniae when there is a T at position 273, a C at positions 488 and 647 and an A at position 653; Proteus mirabilis when there is a C at positions 440 and 488 and a T at position 647; Pseudomonas aeruginosa when there is an A at position 440 and a T at position 647; Streptococcus agalactiae when there is an A at positions 378, 488 and 647; Streptococcus pneumoniae when there is T at positions 488 and 647; and Streptococcus pyogenes when there is G at position 378 and A at positions 488 and 647.
[0076] For example, in another embodiment, bacterium selected from among Acinetobacter calcoaceticus; Enterobacter cloacae; Escherichia coli; Klebsiella pneumoniae; Proteus mirabilis; Pseudomonas aeruginosa; Streptococcus agalactiae; Streptococcus pneumoniae; and Streptococcuspyogenes may be identified in a sample based on the presence of SNPs set forth in the following table:
Table 1 Bacterial SNP position in the 16S rRNA gene as set forth in SEQ ID species NO:1 273 378 412 440 488 647 653 Escherichiacoli T G A C C C T Streptococcuspneumoniae A A A T T T T Streptococcus agalactiae A A A T A A T Streptococcus pyogenes A G A T A A T Proteusmirabilis T G A C C T A Enterobactercloacae T G A C C C G Klebsiella pneumoniae T G A C C C A Pseudomonasaeruginosa A G A A C T A Acinetobacter calcoaceticus A G A A C A A
[0077] For example, in another embodiment, bacterium selected from among Escherichia coli, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Proteus mirabilis, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Enterococcusfaecalis, Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens, Corynebacterium jeikeium, Bacteroides fragilis, Neisseria meningitidis, Haemophilus influenzae, Serratia marcescens, Salmonella sp., and Staphylococcus epidermidis may be identified in a sample based on the presence of SNPs set forth in the following table:
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[0078] In another embodiment, bacterium selected from among Bacillus anthracis, Clostridium botulinum type A, Clostridium botulinum type B, Clostridium botulinum type C, Clostridium botulinum type D, Clostridium botulinum type G, Yersinia pestis, Francisella tularensis, Vibrio cholerae and Burkholderia pseudomallei may be identified in a sample based on SNPs at positions corresponding to positions 746, 764, 771, or 785 of the 16S rRNA gene as set forth in SEQ ID NO:43, wherein the bacterium is: Bacillus anthracis when there is a T at position 746, A at position 764, C at position 771 and G at position 785; Clostridium botulinum type A or Clostridium botulinum type B when there is a T at position 746, G at position 764, C at position 771 and T at position 785; Clostridium botulinum type C when there is a T at position 746, A at position 764, T at position 771 and T at position 785; Clostridium botulinum type D when there is a C at position 746, A at position 764, T at position 771 and T at position 785; Clostridium botulinum type G when there is a T at position 746, G at position 764, C at position 771 and G at position 785; Yersinia pestis when there is a C at position 746, G at position 764, T at position 771 and G at position 785; Francisellatularensis when there is a T at position 746, A at position 764, G at position 771 and G at position 785; Vibrio cholerae when there is a C at position 746, A at position 764, T at position 771 and G at position 785; and Burkholderia pseudomallei when there is a C at position 746, G at position 764, C at position 771 and G at position 785.
[0079] For example, in another embodiment, bacterium selected from among Bacillus anthracis, Clostridium botulinum type A, Clostridium botulinum type B, Clostridium botulinum type C, Clostridium botulinum type D, Clostridium botulinum type G, Yersinia pestis, Francisellatularensis, Vibrio cholerae and Burkholderia pseudomallei may be identified in a sample based on the presence of SNPs set forth in the following table:
Table 3 Bacterial species SNP position in the 16S rRNA gene as set forth in SEQ ID NO: 43 746 764 771 785 Bacillus anthracis T A C G Clostridium botulinum type A T G C T Clostridium botulinum type B T G C T Clostridium botulinum type C T A T T Clostridium botulinum type D C A T T Clostridium botulinum type G T G C G Yersiniapestis C G T G Francisellatularensis T A G G Vibrio cholerae C A T G Burkholderiapseudomallei C G C G
The cumulative discrimatory index of the four SNPs used to identify the above organisms are
0.667 for 1 SNP; 0.889 for 2 SNPs; 0.944 for 3 SNPs; and 0.972 for 4 SNPs.
[0080] Position 746 of the 16S rRNA gene set forth in SEQ ID NO:43 corresponds to position 737 of the 16S rRNA gene set forth in SEQ ID NO:1. Position 764 of the 16S rRNA gene set forth in SEQ ID NO:43 corresponds to position 755 of the 16S rRNA gene set forth in SEQ ID NO:1. Position 771 of the 16S rRNA gene set forth in SEQ ID NO:43 corresponds to position 762 of the 16S rRNA gene set forth in SEQ ID NO:1. Position 785 of the 16S rRNA gene set forth in SEQ ID NO:43 corresponds to position 776 of the 16S rRNA gene set forth in SEQ ID NO:1.
[0081] Therefore, in another embodiment, bacterium selected from among Bacillus anthracis, Clostridium botulinum type A, Clostridium botulinum type B, Clostridium botulinum type C, Clostridium botulinum type D, Clostridium botulinum type G, Yersinia pestis, Francisellatularensis, Vibrio cholerae and Burkholderia pseudomallei may be identified in a sample based on SNPs at positions corresponding to positions 737, 755, 762, or 776 of the 16S rRNA gene as set forth in SEQ ID NO:1, wherein the bacterium is: Bacillus anthracis when there is a T at position 737, A at position 755, C at position 762 and G at position 776; Clostridium botulinum type A or Clostridium botulinum type B when there is a T at position 737, G at position 755, C at position 762 and T at position 776; Clostridium botulinum type C when there is a T at position 737, A at position 755, T at position 762 and T at position 776; Clostridium botulinum type D when there is a C at position 737, A at position 755, T at position 762 and T at position 776; Clostridium botulinum type G when there is a T at position 737, G at position 755, C at position 762 and G at position 776; Yersinia pestis when there is a C at position 737, G at position 755, T at position 762 and G at position 776; Francisellatularensis when there is a T at position 737, A at position 755, G at position 762 and G at position 776; Vibrio cholerae when there is a C at position 737, A at position 755, T at position 762 and G at position 776; and Burkholderia pseudomallei when there is a C at position 737, G at position 755, C at position 762 and G at position 776.
[0082] In another embodiment, yeast organism or filamentous fungi selected from among Candida albicans, Candida tropicalis, Candidaparapsilosis,Candida glabrata,Fusarium spp., Aspergillus fumigatus, and Cryptococcus neoformans may be identified in a sample based on SNPs at positions corresponding to positions 343, 371, 388, 416, and 467 of the 18S rRNA gene set forth in SEQ ID NO: 37, wherein the yeast organism or filamentous fungi is: Candida albicans when there is a C at position 343, A at position 371, T at position 388, G at position 416 and G at position 467; Candida tropicalis when there is a C at position 343, A at position
371, C at position 388, G at position 416 and C at position 467; Candidaparapsilosis when there is a C at position 343, A at position 371, C at position 388, G at position 416 and G at position 467; Candida glabrata when there is a T at position 343, A at position 371, C at position 388, G at position 416 and G at position 467; Fusarium spp. when there is a C at position 343, C at position 371, T at position 388, T at position 416 and G at position 467; Aspergillusfumigatus when there is a C at position 343, C at position 371, T at position 388, C at position 416 and G at position 467; and Cryptococcus neoformans when there is a C at position 343, A at position 371, T at position 388, T at position 416 and G at position 467.
[0083] For example, in another embodiment, yeast organism or filamentous fungi selected from among Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, Fusarium spp., Aspergillus fumigatus, and Cryptococcus neoformans may be identified in a sample based on the presence of SNPs set forth in the following table:
Table 4 Yeast organism or filamentous fungi SNP position in the 18S rRNA gene as set forth in SEQ ID species NO: 37 343 371 388 416 467 Candida albicans C A T G G Candidatropicalis C A C G C Candidaparapsilosis C A C G G Candidaglabrata T A C G G Fusarium sp. C C T T G Aspergillusfumigatus C C T C G Cryptococcus neoformans C A T T G
[0084] The bacteria, yeast organism or filamentous fungi may be partially identified or classified based on one or more of the above SNPs.
[0085] In one embodiment nucleic acid is extracted from the sample prior to analysis in the methods of the invention (especially in the first and second aspects). In another embodiment, the step of analysing in the methods (especially in the first and second aspects) may comprise amplification of nucleic acid. The nucleic acid may be amplified by any method known in the art including, but not limited to polymerase chain reaction (PCR), ligase chain reaction (LCR) and reverse transcription-polymerase chain reaction (RT-PCR) using one or more oligonucleotides/primers that will amplify transcribed RNA.
[0086] The SNPs may be analysed by any method known in the art including, but not limited to: high resolution melt analysis, 5' nuclease digestion (including 5' nuclease digestion), molecular beacons, oligonucleotide ligation, microarray, restriction fragment length polymorphism; antibody detection methods; direct sequencing or any combination thereof. In one embodiment, the step of analysing in the methods (especially in the first and second aspects of the invention) comprises determining the presence or the absence of the at least one SNP using high resolution melt analysis, 5' nuclease digestion, molecular beacons, oligonucleotide ligation, microarray, restriction fragment length polymorphism, antibody detection methods; direct sequencing or any combination thereof. The SNPs may be detected by any method known in the art including, but not limited to: polymerase chain reaction (PCR); ligase chain reaction (LCR); hybridization analysis; high-resolution melt analysis; digestion with nucleases, including 5' nuclease digestion; molecular beacons; oligonucleotide ligations; microarray; restriction fragment length polymorphism; antibody detection methods; direct sequencing; or any combination thereof.
[0087] For example, in some embodiments, the identifying of bacterium or classifying of bacteria may further be based on DNA melting characteristics of the SNPs as broadly described above and their surrounding DNA sequences, preferably high-resolution melt analysis.
[0088] For example, in some such embodiments, the methods of the present invention may further include high-resolution melt (HRM) analysis to further analyse the DNA melting characteristics of the SNPs as broadly described above and their surrounding DNA sequences. In a particular embodiment, the HRM analysis may include forming a DNA amplification product (i.e., amplicon) containing at least one of the SNPs and at least one intercalating fluorescent dye and heating the DNA amplification product through its melting temperature (Tm). The HRM is monitored in real-time using the fluorescent dye incorporated into the DNA amplification product. The level of fluorescence is monitored as the temperature increases with the the fluorescence reducing as the amount of double-stranded DNA reduces. Changes in fluorescence and temperature can be plotted in a graph known as a melt curve.
[0089] As a skilled addressee will understand, the Tm of the DNA amplification product at which the two DNA strands separate is predictable, being dependent on the sequence of the nucleotide bases forming the DNA amplification product. Accordingly, it is possible to differentiate between DNA amplification products including a DNA amplification product containing a polymorphism (i.e., a SNP or SNPs) as the melt curves will appear different. Indeed, in some embodiments, it is possible to differentiate between DNA amplification products containing the same polymorphism based on differences in the surrounding DNA sequences.
[0090] For example, bacterium selected from among Acinetobacter calcoaceticus; Enterobacteraerogenes; Enterobacter cloacae; Enterococcusfaecalis; Enterococcusfaecium;
Escherichiacoli; Klebsiellapneumoniae; Proteusmirabilis;Pseudomonas aeruginosa;Serratia marcescens; Staphylococcus aureus; Staphylococcus epidermidis; Streptococcus agalactiae; Streptococcus pneumoniae; and Streptococcuspyogenes may be identified in a sample based on the presence of SNPs set forth in the following table and DNA melting characteristics of the SNPs and their surrounding DNA sequences:
Table 5 Bacterial SNP position in the 16S rRNA gene as set forth in SEQ ID species NO:1 273 378 412 440 488 647 653 Escherichiacoli T G A C C C T Staphylococcus aureus A G T T C A T Staphylococcus epidermidis A G T T C A T Streptococcuspneumoniae A A A T T T T Streptococcus agalactiae A A A T A A T Streptococcus pyogenes A G A T A A T Enterococcusfaecalis A A A T T G T Enterococcusfaecium A A A T T G T Proteusmirabilis T G A C C T A Serratiamarcescens T G A C T C A Enterobacteraerogenes T G A C T C A Enterobactercloacae T G A C C C G Klebsiella pneumoniae T G A C C C A Pseudomonasaeruginosa A G A A C T A Acinetobacter calcoaceticus A G A A C A A
[0091] For example, bacterium selected from among Escherichia coli, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Proteus mirabilis, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Enterococcus faecalis, Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens, Corynebacterium jeikeium, Bacteroides fragilis, Neisseria meningitidis, Haemophilus influenzae, Serratia marcescens, Salmonella sp., Staphylococcus epidermidis may be identified in a sample based on the presence of SNPs set forth in Table 2 and DNA melting characteristics of the SNPs and their surrounding DNA sequences.
[0092] In one embodiment, bacteria selected from Acinetobacter calcoaceticus; Enterobacteraerogenes; Enterobacter cloacae; Enterococcusfaecalis; Enterococcusfaecium; Escherichiacoli; Klebsiellapneumoniae; Proteusmirabilis;Pseudomonas aeruginosa;Serratia marcescens; Staphylococcus aureus; Staphylococcus epidermidis; Streptococcus agalactiae; Streptococcus pneumoniae; and Streptococcuspyogenes may be identified in a sample based on SNPs at positions corresponding to positions 273, 378, 412, 440, 488, 647 and 653 of the 16S rRNA gene as set forth in SEQ ID NO:1 and high-resolution melt curve analysis of the SNPs and their surrounding DNA.
[0093] For example, the bacterium selected from among Acinetobacter calcoaceticus; Enterobacter cloacae; Escherichia coli; Klebsiella pneumoniae; Proteus mirabilis; Pseudomonas aeruginosa; Streptococcus agalactiae; Streptococcus pneumoniae; and Streptococcus pyogenes may be identified in the sample based on the SNP positions as described above and/or high-resolution melt curve analysis of the SNPs and their surrounding DNA.
[0094] In some embodiments. the bacterium selected from Staphylococcus aureus; Staphylococcus epidermidis; Enterococcus faecalis; Enterococcus faecium; Serratia marcescens; and Enterobacter aerogenes may be individually identified in a sample based on SNPs at positions corresponding to positions 412, 440, 488 and 647 of the 16S rRNA gene as set forth in SEQ ID NO:1, wherein: Staphylococcus aureus and Staphylococcus epidermidis may be identified when there is a T at position 412 and then further distinguished from one another based on high-resolution melt curve analysis of the DNA surrounding the SNP at position 412; Enterococcusfaecalisand Enterococcusfaecium may be identified when there is a G at position 647 and then further distinguished from one another based on high-resolution melt curve analysis of the DNA surrounding the SNP at position 647; Serratia marcescens and Enterobacteraerogenes may be identified when there is a C at positions 440 and 647 and a T at position 488 and may then be further distinguished from one another based on high-resolution melt curve analysis of the DNA surrounding the SNPs at any one of positions 440, 488 and 647.
[0095] In other embodiments, the bacterium selected from Enterococcus faecalis; Enterococcus faecium; Streptococcus agalactiae; and Streptococcus pyogenes may be identified in a sample based on at least one SNP at a position corresponding to position 378 of the 16S rRNA gene as set forth in SEQ ID NO:1 and high-resolution melt curve analysis of the DNA surrounding the SNP at position 378.
[0096] For example, bacterium selected from among Bacillus anthracis, Clostridium botulinum type A, Clostridium botulinum type B, Clostridium botulinum type C, Clostridium botulinum type D, Clostridium botulinum type G, Yersinia pestis, Francisellatularensis, Vibrio cholerae and Burkholderiapseudomallei may be identified in a sample based on the presence of SNPs set forth in the following table and DNA melting characteristics of the SNPs and their surrounding DNA sequences:
Table 6 .actria .pes SNP position in the 16S rRNA gene as set forth in SEQ ID NO: 43 Bacterialspecies 746 764 771 785 Bacillus anthracis T A C G Clostridium botulinum type A T G C T Clostridium botulinum type B T G C T Clostridium botulinum type C T A T T Clostridium botulinum type D C A T T Clostridium botulinum type G T G C G Yersiniapestis C G T G Francisellatularensis T A G G Vibrio cholerae C A T G Burkholderiapseudomallei C G C G
[0097] As noted above, position 746 of the 16S rRNA gene set forth in SEQ ID NO:43 corresponds to position 737 of the 16S rRNA gene set forth in SEQ ID NO:1. Position 764 of the 16S rRNA gene set forth in SEQ ID NO:43 corresponds to position 755 of the 16S rRNA gene set forth in SEQ ID NO:1. Position 771 of the 16S rRNA gene set forth in SEQ ID NO:43 corresponds to position 762 of the 16S rRNA gene set forth in SEQ ID NO:1. Position 785 of the 16S rRNA gene set forth in SEQ ID NO:43 corresponds to position 776 of the 16S rRNA gene set forth in SEQ ID NO:1.
[0098] In one embodiment, bacteria selected from Bacillus anthracis, Clostridium botulinum type A, Clostridium botulinum type B, Clostridium botulinum type C, Clostridium botulinum type D, Clostridium botulinum type G, Yersinia pestis, Francisellatularensis, Vibrio cholerae and Burkholderia pseudomallei may be identified in a sample based on SNPs at positions corresponding to positions 746, 764, 771, or 785 of the 16S rRNA gene as set forth in SEQ ID NO:43 (or positions 737, 755, 762, or 776 of the 16S rRNA gene as set forth in SEQ ID NO:1) and high-resolution melt curve analysis of the SNPs and their surrounding DNA.
[0099] For example, the bacterium selected from among Bacillus anthracis, Clostridium botulinum type A, Clostridium botulinum type B, Clostridium botulinum type C, Clostridium botulinum type D, Clostridium botulinum type G, Yersinia pestis, Francisellatularensis, Vibrio cholerae and Burkholderia pseudomallei may be identified in the sample based on the SNP positions as described above and/or high-resolution melt curve analysis of the SNPs and their surrounding DNA.
[00100] In another example, yeast organism or filamentous fungi selected from among Candida albicans, Candida tropicalis, Candidaparapsilosis,Candida glabrata,Fusarium spp., Aspergillus fumigatus, and Cryptococcus neoformans may be identified in a sample based on the presence of SNPs set forth in the following table and DNA melting characteristics of the
SNPs and their surrounding DNA sequences:
Table 7 Yeast organism or filamentous fungi SNP position in the 18S rRNA gene as set forth in SEQ ID species NO: 37 343 371 388 416 467 Candida albicans C A T G G Candidatropicalis C A C G C Candidaparapsilosis C A C G G Candidaglabrata T A C G G Fusarium sp. C C T T G Aspergillusfumigatus C C T C G Cryptococcus neoformans C A T T G
[00101] In one embodiment, yeast organism or filamentous fungi selected from Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, Fusarium spp., Aspergillus fumigatus, and Cryptococcus neoformans may be identified in a sample based on SNPs at positions corresponding to positions 343, 371, 388, 416, and 467 of the 18S rRNA gene set forth in SEQ ID NO: 37 and high-resolution melt curve analysis of the SNPs and their surrounding DNA.
[00102] For example, the yeast organism or filamentous fungi selected from among Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, Fusarium spp., Aspergillusfumigatus, and Cryptococcus neoformans may be identified in the sample based on the SNP positions as described above and/or high-resolution melt curve analysis of the SNPs and their surrounding DNA.
[00103] In some embodiments, nucleic acid may be extracted from the sample prior to analysis, identifying, classifying and/or diagnosing. Generally, the analysis, identifying, classifying and/or diagnosing may include amplification of the nucleic acid. In some embodiments, the analysis, identifying, classifying and/or diagnosing may further include administering a therapeutic agent to the subject, such as, e.g., an antibiotic. In another embodiment, a method of treatment (for example as in the third aspect of the present invention) may further comprise the step of determining whether the at least one bacteria, yeast organism or filamentous fungi is resistant to a therapeutic agent.
[00104] In one embodiment, any suitable sample may be used in the methods of the present invention. Exemplary samples may comprise sputum, saliva, blood, cerebrospinal fluid or urine samples.
[00105] The probe, tool or reagent may be, but is not limited to, an oligonucleotide, a primer, a nucleic acid, a polynucleotide, DNA, cDNA, RNA, a peptide or a polypeptide. These may be, for example, single stranded or double stranded, naturally occurring, isolated, purified, chemically modified, recombinant or synthetic.
[00106] The probe, tool or reagent may be, but is not limited to, an antibody or other type of molecule or chemical entity capable of specifically binding, detecting or identifying at least a portion of a 16S rRNA gene or an 18S rRNA gene in a sample containing at least one SNP.
[00107] The probe, tool or reagent may be any number or combination of the above, and the number and combination will depend on a desired result to be achieved - e.g., detection of SNP at a genomic level (genotyping) or at the RNA transcription level.
[00108] The probe, tool or reagent may be isolated. The probe, tool or reagent may be detectably labelled. A detectable label may be included in an amplification reaction. Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2',7'-dimethoxy-4',5' dichloro-6-carboxyfluorescein (JOE), 6-carboxy-X-rhodamine (ROX), 6-carboxy-2',4',7',4,7 hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N',N'-tetramethyl-6 carboxyrhodamine (TAMRA), radioactive labels, e.g. 32 35, 3H; etc. The label may be a two stage system, where the amplified DNA is conjugated to biotin, haptens, etc. having a high affinity binding partner, e.g. avidin, specific antibodies, etc., where the binding partner is conjugated to a detectable label. The label may be conjugated to one or both of the primers. Alternatively, the pool of nucleotides used in the amplification is labeled, so as to incorporate the label into the amplification product.
[00109] In particular embodiments, the at least one probe, tool or reagent is for specifically binding, detecting or identifying of a SNP at the genomic level or transcription level, preferably the former.
[00110] In preferred embodiments, the at least one probe, tool or reagent is for specifically binding, detecting or identifying at least a portion of a 16S rRNA or a 18S rRNA gene or gene product in a sample containing at least one SNP as set forth in any one of Tables 1 to 7.
[00111] When identifying, partially identifying, classifying a bacterium, yeast organism or filamentous fungi, or identifying, partially identifying or diagnosing a bacterial, yeast organism or filamentous fungi infection a single probe (especially primer) may be used with each sample, or multiple probes (especially primers) may be used with each sample (i.e. in one pot). Such probes (especially primers) can be added to the raw solution obtained from amplification (such as PCR).
[00112] In one embodiment, the at least one probe, tool or reagent may comprise two primers, each of which hybridizes to at least a portion of a bacterial 16S rRNA gene (or gene product) or to at least a portion of a yeast organism or filamentous fungi 18S rRNA gene (or gene product), containing a SNP as defined above. In another embodiment, the at least one probe, tool or reagent may comprise a probe that hybridizes to at least a portion of a bacterial 16S rRNA gene or gene product or to at least a portion of a yeast organism or filamentous fungi 18S rRNA gene or gene product containing a SNP as defined above.
[00113] In one embodiment, said at least one probe, tool or reagent comprises an oligonucleotide having (or comprising or consisting of) a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence homology or identity with the sequence as set forth in at least one of SEQ ID Nos 16-35 and 53-57. Said probe, tool or reagent may be a primer. Said probe, tool or reagent may comprise an oligonucleotide having a nucleotide sequence as set forth in at least one of SEQ ID NOs: 16-35 and 53-57.
[00114] In one aspect, the present invention provides at least one isolated probe, tool or reagent, wherein said at least one isolated probe, tool or reagent comprises an oligonucleotide having (or comprising or consisting of) a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence homology or identity with the sequence as set forth in at least one of SEQ ID Nos 16-35 and 53-57. Said probe, tool or reagent may be a primer.
[00115] Suitable primers for identification of SNPs in the 16S rRNA sequence set forth in SEQ ID NO. 43 (especially for identification of the SNPs in Table 3) may be as shown in the Table below.
Table 8 Forward primer sequence Reverse primer sequence 5'GTGTAGCGGTGAAATGCGTAGAG 3' 5'TCGTTTACCGTGGACTACCAGGG 3' (SEQ (SEQ ID NO.56) ID NO. 57)
[00116] Suitable primers for identification of SNPs in the 18S rRNA sequence set forth in SEQ ID NO. 37 (especially for identification of the SNPs in Table 4) may be as shown in the Table below.
Table 9 Forward primer sequence Reverse primer sequence 5'CATCCAAGGAAGGCAGCAGGCGCG3' 5'GTTCAACTACGAGCTTTTTAAC 3' (SEQ (SEQ ID NO, 53) ID NO. 54) 5'GTTCGACTACGAGCTTTTTAAC 3' (SEQ ID NO. 55)
[00117] Any of the features described herein can be combined in any combination with any one or more of the other features described herein within the scope of the invention.
[00118] The above largely discusses the use of 16S rRNA and 18S rRNA genes. However, the above may also be applicable to 16S rRNA and to 18S rRNA and to other 16S rRNA and 18S rRNA gene products. Accordingly in some embodiments (and where appropriate), references to 16S rRNA gene and 18S rRNA gene above and below may be replaced with 16S rRNA gene product (or 16S rRNA) and 18S rRNA gene product (or 18S rRNA).
[00119] The reference to any prior art in this specification is not, and should not be taken as an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge.
BRIEF DESCRIPTION OF DRAWINGS
[00120] Various embodiments of the invention will be described with reference to the following drawings, in which:
[00121] Figure 1 is a CLUSTALW sequence alignment of the representative genes encoding 16S rRNA molecules from the following bacterial species: Acinetobacter calcoaceticus; Enterobacteraerogenes; Enterobacter cloacae; Enterococcusfaecalis; Enterococcusfaecium; Escherichiacoli; Klebsiellapneumoniae; Proteusmirabilis;Pseudomonas aeruginosa;Serratia marcescens; Staphylococcus aureus; Staphylococcus epidermidis; Streptococcus agalactiae; Streptococcus pneumoniae; and Streptococcuspyogenes. Variable sequences, as determined by the CLUSTALW alignment were removed. The SNPs at positions corresponding to positions 273, 378, 412, 440, 488, 647 and 653 of the 16S rRNA gene from E. coli as set forth in SEQ ID NO:1 are highlighted together with the corresponding nucleotide in the aligned sequences.
[00122] Figure 2 shows a normalised high-resolution melt (HRM) curves plot for the following 15 bacterial species tested in Example 1: Acinetobacter calcoaceticus; Enterobacter aerogenes; Enterobacter cloacae; Enterococcus faecalis; Enterococcusfaecium; Escherichia coli; Klebsiella pneumoniae; Proteus mirabilis; Pseudomonas aeruginosa; Serratia marcescens; Staphylococcus aureus; Staphylococcus epidermidis; Streptococcus agalactiae;
Streptococcus pneumoniae; and Streptococcus pyogenes.
[00123] Figure 3 shows a difference HRM curves plot for the 15 bacterial species shown in Figure 2 with Escherichiacoli used as a baseline.
[00124] Figure 4 shows a normalised HRM curves plot for Staphylococcus aureus and Staphylococcus epidermidis for amplicons containing a SNP at a position corresponding to position 412 in the 16S rRNA gene as set forth in SEQ ID NO:1.
[00125] Figure 5 shows a difference HRM curves plot for Staphylococcus aureus and Staphylococcus epidermidis as shown in Figure 4 with S. aureus used as the baseline.
[00126] Figure 6 shows a normalised HRM curves plot for Enterococcusfaecalis, Enterococcusfaecium, Streptococcus agalactiae;Streptococcus pneumoniae and Streptococcus pyogenes for amplicons containing a SNP at a position corresponding to position 378 in the 16S rRNA gene as set forth in SEQ ID NO:1.
[00127] Figure 7 shows a difference HRM curves plot for Enterococcus faecalis, Enterococcusfaecium, Streptococcus agalactiae;Streptococcus pneumoniae and Streptococcus pyogenes as shown in Figure 6 with E.faecalis used as the baseline.
[00128] Figure 8 shows a normalised HRM curves plot for Escherichia coli, Enterobacter cloacae, Serratia marcescens, Acinetobacter calcoaceticus, Enterobacter aerogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae and Proteus mirabilis for amplicons containing a SNP at a position corresponding to position 412 in the 16S rRNA gene as set forth in SEQ ID NO:1.
[00129] Figure 9 shows a difference HRM curves plot for Escherichia coli, Enterobacter cloacae, Serratia marcescens, Acinetobacter calcoaceticus, Enterobacter aerogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae and Proteus mirabilis as shown in Figure 8 with Escherichiacoli used as the baseline.
[00130] Figure 10 shows a normalised HRM curves plot for Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes for amplicons containing a SNP at a position corresponding to position 378 in the 16S rRNA gene as set forth in SEQ ID NO:1.
[00131] Figure 11 shows a difference HRM curves plot for Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes as shown in Figure 10 with Streptococcus pneumoniae used as the baseline.
[00132] Figure 12 shows a normalised HRM curves plot for Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, Enterobacter cloacae, and Serratia marcescens for amplicons containing a SNP at a position corresponding to position 412 in the 16S rRNA gene as set forth in SEQ ID NO:1.
[00133] Figure 13 shows a normalised HRM curves plot for Streptococcus pneumonaie, Streptococcus agalactiae and Streptococcus pyogenes for amplicons containing a SNP at a position corresponding to position 412 in the 16S rRNA gene as set forth in SEQ ID NO:1.
KEY TO SEQUENCE LISTING
[00134] SEQ ID NO:1: Escherichia coli 16S rRNA gene (Genbank accession NR_102804.1);
[00135] SEQ ID NO:2: Staphylococcus aureus 16S rRNA gene (Genbank accession NR_075000.1);
[00136] SEQ ID NO:3: Staphylococcus epidermidis 16S rRNA gene (Genbank accession NR_074995.1);
[00137] SEQ ID NO:4: Streptococcus pneumoniae 16S rRNA gene (Genbank accession NR_074564.1);
[00138] SEQ ID NO:5: Streptococcus agalactiae 16S rRNA gene (Genbank accession NR_040821.1);
[00139] SEQ ID NO:6: Streptococcus pyogenes 16S rRNA gene (Genbank accession NR_074091.1);
[00140] SEQ ID NO:7: Enterococcus faecalis 16S rRNA gene (Genbank accession NR_074637.1);
[00141] SEQ ID NO:8: Enterococcus faecium 16S rRNA gene (Genbank accession NR_042054.1);
[00142] SEQ ID NO:9: Proteus mirabilis 16S rRNA gene (Genbank accession NR_074898.1);
[00143] SEQ ID NO:10: Serratia marcescens 16S rRNA gene (Genbank accession NR_041980.1);
[00144] SEQ ID NO:11: Enterobacter aerogenes 16S rRNA gene (Genbank accession NR_024643.1);
[00145] SEQ ID NO:12: Enterobacter cloacae 16S rRNA gene (Genbank accession
Received 03/06/2019 35
NR_028912.1);
[00146] SEQ ID NO:13: Klebsiella pneunoniae 16S rRNA gene (Genbank accession NR_036794.1);
[00147] SEQ ID NO:14: Pseudoinonas aeruginosa 16S rRNA gene (Genbank accession NR_074828.1);
[00148] SEQ ID NO:15: Acinetobacter calcoaceticus 16S rRNA gene (Genbank accession AB302132.1);
[00149] SEQ ID NO:16: Forward Primer (CCTCTTGCCATCGGATGTG);
[00150] SEQ ID NO:17: Reverse Primer (CCAGTGTGGCTGGTCATCCT);
[00151] SEQ ID NO:18: Forward Primer (CCTACGGGAGGCAGCAGTAG);
[00152] SEQ ID NO:19: Forward Primer (GGGAGGCAGCAGTAGGGAAT);
[00153] SEQ ID NO:20: Reverse Primer (CGATCCGAAAACCTTCTTCACT);
[00154] SEQ ID NO:21: Forward Primer (AAGACGGTCTTGCTGTCACTTATAGA);
[00155] SEQ ID NO:22: Reverse Primer (CTATGCATCGTTGCCTTGGTAA);
[00156] SEQ ID NO:23: Forward Primer (TGCCGCGTGAATGAAGAA);
[00157] SEQ ID NO:24: Forward Primer (GCGTGAAGGATGAAGGCTCTA);
[00158] SEQ ID NO:25: Forward Primer (TGATGAAGGTTTTCGGATCGT);
[00159] SEQ ID NO:26: Reverse Primer (TGATGTACTATTAACACATCAACCTTCCT);
[00160] SEQ ID NO:27: Reverse Primer (AACGCTCGGATCTTCCGTATTA);
[00161] SEQ ID NO:28: Reverse Primer (CGCTCGCCACCTACGTATTAC);
[00162] SEQ ID NO:29: Forward Primer (GTTGTAAGAGAAGAACGAGTGTGAGAGT);
[00163] SEQ ID NO:30: Reverse Primer (CGTAGTTAGCCGTCCCTTTCTG);
[00164] SEQ ID NO:31: Forward Primer (GCGGTTTGTTAAGTCAGATGTGAA);
[00165] SEQ ID NO:32: Forward Primer (GGTCTGTCAAGTCGGATGTGAA);
[00166] SEQ ID NO:33: Forward Primer (TCAACCTGGGAACTCATTCGA);
[00167] SEQ ID NO:34: Reverse Primer (GGAATTCTACCCCCCTCTACGA);
[00168] SEQ ID NO:35: Reverse Primer (GGAATTCTACCCCCCTCTACAAG);
AMFNnFT I-IFFT
[00169] SEQ ID NO:36: Aspergillus fumigatus strain MJ-X6 18S ribosomal RNA gene, complete sequence (GenBank accession HM590663.1);
[00170] SEQ ID NO:37: Candida albicans 18S ribosomal RNA gene, complete sequence (GenBank accession AF114470.1);
[00171] SEQ ID NO:38: Candida glabrata strain SZ2 18S ribosomal RNA gene, partial sequence (GenBank accession KT229542.1);
[00172] SEQ ID NO:39: Candida parapsilosis 18S ribosomal RNA gene, partial sequence (GenBank accession DQ218328.1);
[00173] SEQ ID NO:40: Candida tropicalis 18S ribosomal RNA genes, partial sequence (GenBank accession AH009771.2);
[00174] SEQ ID NO:41: Cryptococcus neoformans var. grubii H99 18S ribosomal RNA rRNA (GenBank accession / NCBI Reference Sequence: XR_001045463.1);
[00175] SEQ ID NO:42: Fusarium sp. strain Z10 18S ribosomal RNA gene, partial sequence (GenBank accession MF973465.1);
[00176] SEQ ID NO:43: Bacillus anthracis strain 2000031664 16S ribosomal RNA gene, partial sequence (GenBank accession AY138383.1);
[00177] SEQ ID NO:44: Burkholderiapseudomallei 16S rRNA gene (GenBank accession AJ131790.1);
[00178] SEQ ID NO:45: Clostridium botulinum type A rrn gene for 16S RNA (GenBank accession X68185.1);
[00179] SEQ ID NO:46: Clostridium botulinum type B rrn gene for 16S RNA (GenBank accession X68186.1);
[00180] SEQ ID NO:47: Clostridium botulinum type C rrn gene for 16S rRNA (GenBank accession X68315.1);
[00181] SEQ ID NO:48: Clostridium botulinum type D rrn gene for 16S RNA (GenBank accession X68187.1);
[00182] SEQ ID NO:49: Clostridium botulinum type G rrn gene for 16S rRNA (GenBank accession X68317.1);
[00183] SEQ ID NO:50: Francisella tularensis strain B-38 16S ribosomal RNA, partial sequence (GenBank accession / NCBI Reference Sequence: NR_029362.1);
[00184] SEQ ID NO:51: Vibrio cholerae strain DL2 16S ribosomal RNA gene, partial sequence (GenBank accession MG062858.1);
[00185] SEQ ID NO:52: Yersinia pestis 16S rRNA gene, isolate: SS-Yp-116 (GenBank accession AJ232238.1);
[00186] SEQ ID NO:53: Forward Primer (CATCCAAGGAAGGCAGCAGGCGCG);
[00187] SEQ ID NO:54: Reverse Primer (GTTCAACTACGAGCTTTTTAAC);
[00188] SEQ ID NO:55: Reverse Primer (GTTCGACTACGAGCTTTTTAAC);
[00189] SEQ ID NO:56: Forward Primer (GTGTAGCGGTGAAATGCGTAGAG);
[00190] SEQ ID NO:57: Reverse Primer (TCGTTTACCGTGGACTACCAGGG).
DETAILED DESCRIPTION
1. Definitions
[00191] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.
[00192] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical objection of the article. By way of example, "an element" means one element or more than one element.
[00193] "Amplification product" or "amplicon" refers to a nucleic acid product generated by nucleic acid amplification techniques.
[00194] The term "biological sample" as used herein refers to a sample that may be extracted, untreated, treated, diluted or concentrated from a patient or subject. Suitably, the biological sample is selected from any part of a patient or subject's body, including, but not limited to, hair, skin, nails, tissues or bodily fluids such as sputum, saliva, cerebrospinal fluid, urine and blood.
[00195] In the present specification and claims (if any), the word "comprising" and its derivatives including "comprises" and "comprise" include each of the stated integers but does not exclude the inclusion of one or more further integers.
[00196] As used herein, "corresponding" nucleic acid positions or nucleotides refer to positions or nucleotides that occur at aligned loci of two or more nucleic acid molecules. Related or variant polynucleotides can be aligned by any method known to those of skill in the art. Such methods typically maximise matches, and include methods such as using manual alignments and by using the numerous alignment programs available (e.g., BLASTN) and others known to those of skill in the art. By aligning the sequences of polynucleotides, one skilled in the art can identify corresponding nucleotides or positions using identical nucleotides as guides. For example, by aligning sequences of the gene encoding the E. coli 16S rRNA (set forth in SEQ ID NO:1) with a gene encoding a 16S rRNA from another species, one of skill in the art can identify corresponding positions and nucleotides using conserved nucleotides as guides.
[00197] By "gene" is meant a unit of inheritance that occupies a specific locus on a genome and consists of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e., introns, 5' and 3' untranslated sequences).
[00198] By "gene product" is meant a product of the gene. For example, a gene product of the 16S rRNA gene includes 16S rRNA. Similarly, a gene product of the 18S rRNA gene includes 18S rRNA. Gene products also include, for example, cDNA sequences derived from the rRNA sequences. Gene products may also include products of the rRNA in which a SNP in the rRNA gene would result in a corresponding change in the product.
[00199] "Homology" refers to the percentage number of nucleic acids or amino acids that are identical or constitute conservative substitutions. Homology can be determined using sequence comparison programs such as GAP (Deveraux et al. 1984), which is incorporated herein by reference. In this way, sequences of a similar or substantially different length to those cited herein could be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.
[00200] "Hybridization" is used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid. In DNA, A pairs with T and C pairs with G. In RNA, U pairs with A and C pairs with G. In this regard, the terms "match" and "mismatch" as used herein refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that do not hybridize efficiently. The nucleotide symbols are set forth in the following table:
Table 10 - Nucleotide Symbols Symbol Description A Adenosine C Cytidine G Guanosine T Thymidine U Uridine M Amino (adenosine, cytosine) K Keto (guanosine, thymidine) R Purine (adenosine, guanosine) Y Pyrimidine (cytosine, thymidine) N Any nucleotide
[00201] By "isolated" is meant material that is substantially or essentially free from components that normally accompany it in its native state.
[00202] The term "oligonucleotide" as used herein refers to a polymer composed of a multiplicity of nucleotide residues (deoxynucleotides or ribonucleotides, or related structural
variants or synthetic analogues thereof) linked via phosphodiester bonds (or related structural
variants or synthetic analogues thereof). Thus, while the term "oligonucleotide" typically refers to a nucleotide polymer in which the nucleotide residues and linkages between them are
naturally occurring, it will be understood that the term also includes within its scope various
analogues including, but not restricted to, peptide nucleic acids (PNAs), phosphoramidates,
phosphorothioates, methyl phosphonates, 2-0-methyl ribonucleic acids, and the like. The exact
size of the molecule can vary depending on the particular application. An oligonucleotide is
typically rather short in length generally from about 10 to 30 nucleotide residues, but the term
can refer to molecules of any length, although the term "polynucleotide" or "nucleic acid" is typically used for large oligonucleotides.
[00203] The terms "patient" and "subject" are used interchangeably and refer to patients and subjects of human or other mammal and includes any individual being examined or treated
using the methods of the invention. However, it will be understood that "patient" does not imply that symptoms are present. Suitable mammals that fall within the scope of the invention
include, but are not restricted to, primates, livestock animals (e.g., sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters),
companion animals (e.g., cats, dogs) and captive wild animals (e.g., koalas, bears, wild cats,
wild dogs, wolves, dingoes, foxes and the like).
[00204] The term "polymorphism" as used herein refers to a difference in the nucleotide or amino acid sequence of a given region as compared to a nucleotide or amino acid sequence in a homologous-region of another individual, in particular, a difference in the nucleotide or amino acid sequence of a given region which differs between individuals of the same species. A polymorphism is generally defined in relation to a reference sequence. Polymorphisms include single nucleotide differences, differences in more than one nucleotide, and single or multiple nucleotide insertions, inversions and deletions; as well as single amino acid differences, differences in sequence of more than one amino acid, and single or multiple amino acid insertions, inversions and deletions. A "polymorphic site" is the locus at which variation occurs. It shall be understood that where a polymorphism is present in a nucleic acid sequence, and reference is made to the presence of a particular base or bases at a polymorphic site, the present invention encompasses the complementary base or bases on the complementary strand at that site.
[00205] The term "polynucleotide" or "nucleic acid" as used herein designates mRNA, RNA, rRNA, cRNA, cDNA, or DNA. The term typically refers to oligonucleotides greater than 30 nucleotides residues in length.
[00206] By "primer" it is meant an oligonucleotide which, when paired with a strand of DNA, is capable of initiating the synthesis of a primer extension product in the presence of a suitable polymerizing agent. The primer is preferably a single-stranded for maximum efficiency in amplification but can alternatively be double-stranded. A primer must be sufficiently long to prime the synthesis of extension products in the presence of the polymerization agent. The length of the primer depends on many factors, including application, temperature to be employed, template reaction conditions, other reagents, and source of primers. For example, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15 to 35 or more nucleotide residues, although it can contain fewer nucleotide residues. Primers can be large polynucleotides, such as from about 200 nucleotides to several kilobases or more. Primers can be selected to be "substantially complementary" to the sequence on the template to which it is designed to hybridize and serve as a site for the initiation of synthesis. By "substantially complementary" it is meant that the primer is sufficiently complementary to hybridize with a target polynucleotide. In some embodiments, the primer contains no mismatches with the template to which it is designed to hybridize but this is not essential. For example, non-complementary nucleotide residues can be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the template. Alternatively, non-complementary nucleotide residues or a stretch of non complementary nucleotide residues can be interspersed into a primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize therewith and thereby form a template for synthesis of the extension product of the primer.
[00207] "Probe" refers to a molecule that binds to a specific sequence or sub-sequence or other moiety of another molecule. Unless otherwise indicated, the term "probe" typically refers to a polynucleotide probe that binds to another polynucleotide, often called the "target polynucleotide", through complementary base pairing. Probes can bind target polynucleotides lacking complete sequence complementarity with the probe, depending on the stringency of the hybridization conditions. Probes can be labelled directly or indirectly.
[00208] The term "sepsis" is used herein in accordance with its normal meaning in clinical medicine, and includes, for example systemic and/or blood-borne infections, such as bacterial infections.
[00209] The term "sepsis-associated bacteria" refers to bacteria that have been identified as being able to cause sepsis in a subject, or have been identified in the blood of a subject with sepsis. "Mammalian (e.g., human) sepsis-associated bacteria" therefore refers to bacteria that have been identified as being able to cause sepsis in a mammalian (e.g., human) subject, or have been identified in the blood of a mammalian (e.g., human) subject with sepsis. Examples of mammalian (e.g., human) sepsis-associated bacteria include Acinetobacter baumannii, Actinobacillus hominis, Actinomyces massiliensis, Aeromonas hydrophila, Bacillus anthracis, Bacteroides fragilis, Brucella abortus, Burkholderia cepacia, Campylobacter coli, Campylobacterfetus, Campylobacter jejuni, Campylobacter lari, Cardiobacteriumvalvarum, Chlamydia trachomatis, Chlamydophila abortus, Chlamydophila pneumoniae, Citrobacter freundii, Clostridium difficile, Clostridium perfringens, Corynebacterium diphtheriae, Corynebacterium jeikeium, Corynebacterium urealyticum, Dermatophilus congolensis, Edwardsiella tarda, Enterobacter aerogenes, Enterobacter cloacae, Enterococcusfaecalis, Enterococcusfaecium, Erysipelothrix rhusiopathiae,Escherichia coli, Eubacterium desmolans, Flavobacterium ceti, Haemophilus ducreyi, Haemophilus influenzae, Haemophilus parahaemolyticus, Haemophilus parainfluenzae, Helicobacter cinaedi, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumonia, Lactobacillus intestinalis, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Micrococcus luteus, Mobiluncus curtisii, Moraxella catarrhalis, Morganella morganii, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroids, Nocardia brasiliensis, Pasteurella multocida, Peptostreptococcus stomatis, Porphyromonas gingivalis, Prevotella buccae, Prevotella intermedia, Prevotella melaninogenica, Proteus mirabilis, Providencia alcalifaciens, Pseudomonas aeruginosa, Rhodococcus equi, Salmonella enterica, Serratia marcescens, Shigella dysenteriae, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, Stenotrophomonas maltophila, Streptococcus agalactiae, Streptococcus anginosus, Streptococcus bovis, Streptococcus constellatus, Streptococcus dysgalactiae, Streptococcus intermedins, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus sanguinis, Streptococcus sobrinus, Streptomyces anulatus, Streptomyces somaliensis, Veillonella atypica, Veillonella denticariosi, Veillonella dispar, Veillonella parvula, Veillonella rogosae, Vibrio cholerae, Yersinia enterocolitica and Yersinia pestis.
[00210] As used herein, "sepsis" is defined as SIRS with a presumed or confirmed infectious process. Confirmation of infectious process can be determined using microbiological culture or isolation of the infectious agent. From an immunological perspective, sepsis may be seen as a systemic response to microorganisms or systemic infection.
[00211] "Systemic Inflammatory Response Syndrome (SIRS)," as used herein, refers to a clinical response arising from a non-specific insult with two or more of the following measureable clinical characteristics; a body temperature greater than 38°C or less than 36°C, a heart rate greater than 90 beats per minute, a respiratory rate greater than 20 per minute, a white 3 3 blood cell count (total leukocytes) greater than 12,000 per mm or less than 4,000 per mm , or a band neutrophil percentage greater than 10%. From an immunological perspective, it may be seen as representing a systemic response to insult (e.g., major surgery) or systemic inflammation. As used herein, therefore, "infection-negative SIRS (inSIRS)" includes the clinical response noted above but in the absence of an identifiable infectious process.
[00212] The term "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which identical nucleic acid base (e.g., A, T, C, G) occurs in both sequence to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity (% seq. identity).
[00213] As used herein, the term single nucleotide polymorphism (SNP) refers to nucleotide sequence variations that occur when a single nucleotide (A, T, C or G) in the genome sequence is altered (such as via substitutions, addition or deletion). SNPs can occur in both coding (gene) and noncoding regions of the genome such as the genome of a prokaryotic or eukaryotic microorganism.
[00214] As used herein, the terms "treatment", "treating" and the like, refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing an infection, condition or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for an infection, condition and/or adverse affect attributable to the infection or condition. "Treatment" as used herein covers any treatment of an infection or condition in a mammal (e.g., a human), and includes: (a) inhibiting the infection or condition, i.e., arresting its development; and (b) relieving the infection or condition, i.e., causing regression of the infection or condition.
2. Polymorphisms of the Invention
[00215] The present invention is based in part on the determination that SNPs within the 16S rRNA gene (and thus within the 16S rRNA molecule) of bacteria can be used to identify individual species of bacterium and/or classify bacteria based on genus or as Gram-positive or Gram-negative. The present invention is also based in part on the determination that SNPs within the 18S rRNA gene (and thus within the 18S rRNA molecule) can be used to identify, partially identify or classify yeast organisms or filamentous fungi.
2.1 Classification of bacteria using SNPs in 16S rRNA
[00216] The present invention provides methods for classifying bacterial species based on genus as well as methods for determining the Gram status of bacteria in a sample, i.e., determining whether the bacteria are Gram-positive or Gram-negative.
[00217] As demonstrated herein, polymorphisms at nucleotide positions of the gene encoding 16S rRNA (and thus of the 16S rRNA molecule itself) that correspond to positions 273 and 653 of the E. coli 16S rRNA gene as set forth in SEQ ID NO:1 can be used to determine the gram status of a selection of bacterial species within a sample, particularly including mammalian (e.g., human) pathogens (including the most commonly found bacterial species isolated by blood culture (Karlowsky et al.2004)).
[00218] Most particularly and as shown in Figure 1, the present invention provides methods for classifying bacterial species selected from among: Acinetobacter calcoaceticus; Enterobacteraerogenes; Enterobacter cloacae; Enterococcusfaecalis; Enterococcusfaecium; Escherichiacoli; Klebsiellapneumoniae; Proteusmirabilis;Pseudomonas aeruginosa;Serratia marcescens; Staphylococcus aureus; Staphylococcus epidermidis; Streptococcus agalactiae; Streptococcus pneumoniae; and Streptococcus pyogenes.
[00219] For example, the method for determining the Gram status of one of the bacterium listed in the above paragraph in a sample includes analysing nucleic acid from the sample for SNPs in the 16S rRNA gene (or 16S rRNA or DNA copy thereof) at positions corresponding to positions 273 and 653 of the 16S rRNA gene set forth in SEQ ID NO:1, wherein an A at position 273 and a T at position 653 indicates that the bacterium in the sample is Gram-positive.
[00220] Another method for determining the Gram status of one of the above said bacterium in a sample, includes analysing nucleic acid from the sample for SNPs in the 16S rRNA gene (or 16S rRNA or DNA copy thereof) at position 440 of the 16S rRNA gene set forth in SEQ ID NO:1, wherein a T at position 440 indicates that the bacterium in the sample is Gram-positive.
[00221] The method for classifying at least one of the above said bacterium in a sample as belonging to a particular genus includes analysing nucleic acid from the sample for SNPs in the 16S rRNA gene (or 16S rRNA or DNA copy thereof) at positions corresponding to positions 412 and 647 of the 16S rRNA gene set forth in SEQ ID NO:1, wherein: a T at position 412 indicates that the bacterium belongs to Staphylococcus genus; or a G at position 647 indicates that the bacterium belongs to the Enterococcus genus.
2.2 Identification of bacteria using SNPs in 16S rRNA and yeast organisms andfilamentous fungi using 18S rRNA
[00222] The present invention also provides methods for identifying bacterium in a sample.
[00223] As demonstrated herein, polymorphisms at nucleotide positions of the gene encoding 16S rRNA (and thus of the 16S rRNA molecule itself) that correspond to any one of positions 273, 378, 412, 440, 488, 647 and 653 of the E. coli 16S rRNA gene as set forth in SEQ ID NO:1 can be used to identify bacterium within a sample, particularly including mammalian (e.g., human) pathogens (including the most commonly found bacterial species isolated by blood culture (Karlowsky et al.2004)).
[00224] In one embodiment, and as shown in Figure 1, the present invention provides methods for identifying bacterium selected from among: Acinetobacter calcoaceticus; Enterobacteraerogenes; Enterobacter cloacae; Enterococcusfaecalis; Enterococcusfaecium; Escherichiacoli; Klebsiellapneumoniae; Proteusmirabilis;Pseudomonas aeruginosa;Serratia marcescens; Staphylococcus aureus; Staphylococcus epidermidis; Streptococcus agalactiae; Streptococcus pneumoniae; and Streptococcus pyogenes.
[00225] The general rules for identifying the above bacterial species within a sample using the above SNPs are depicted in Table 1 and/or Table 5.
[00226] From the above bacteria, the bacterium Enterobactercloacae can be identified in a sample by analysing nucleic acid from the sample for an SNP in the 16S rRNA gene (or 16S rRNA or DNA copy thereof) at a position corresponding to position 653 of the 16S rRNA gene set forth in SEQ ID NO:1, wherein a G at position 653 indicates that the bacterium is Enterobactercloacae.
[00227] From the above bacteria, bacterium selected from Streptococcus agalactiae, Streptococcus pneumoniae and Streptococcus pyogenes can be identified in a sample by analysing nucleic acid from the sample for SNPs in the 16S rRNA gene (or 16S rRNA or DNA copy thereof) at positions corresponding to positions 378 and 488 of the 16S rRNA gene set forth in SEQ ID NO:1, wherein: an A at position 378 and a T at position 488 indicate that the bacterium is Streptococcus pneumoniae; an A at positions 378 and 488 indicate that the bacterium is Streptococcus agalactiae; and a G at position 378 and an A at position 488 indicate that the bacterium is Streptococcuspyogenes.
[00228] From the above bacteria, the method can identify bacterium selected from Acinetobacter calcoaceticus; Enterobacter cloacae; Escherichia coli; Klebsiella pneumoniae; Proteus mirabilis; Pseudomonas aeruginosa; Streptococcus agalactiae; Streptococcus pneumoniae; and Streptococcus pyogenes in a sample by analysing nucleic acid from the sample for SNPs in the 16S rRNA gene (or 16S rRNA or DNA copy thereof) at positions corresponding to positions 273, 378, 412, 440, 488, 647 and 653 of the 16S rRNA gene set forth in SEQ ID NO:1, wherein: an A at positions 273, 440 and 647 indicates that the bacterium is Acinetobacter calcoaceticus; a G at position 653 indicates that the bacterium is Enterobacter cloacae; a T at positions 273 and 653 indicates that the bacterium is E. coli; a T at position 273, a C at positions 488 and 647 and an A at position 653 indicates that the bacterium is Klebsiella pneumoniae; a C at positions 440 and 488 and a T at position 647 indicates that the bacterium is Proteus mirabilis; an A at position 440 and a T at position 647 indicates that the bacterium is Pseudomonas aeruginosa; an A at positions 378, 488 and 647 indicates that the bacterium is Streptococcus agalactiae; a T at positions 488 and 647 indicates that the bacterium is Streptococcus pneumoniae; and a G at position 378 and an A at positions 488 and 647 indicates that the bacterium is Streptococcus pyogenes.
[00229] In addition to the above, the methods of the present invention can also be used to identify the presence of the following bacterium in a sample: Staphylococcus aureus; S.
epidermidis; Enterococcus faecalis; Enterococcus faecium; Serratia marcescens; and Enterobacteraerogenes. The methods again include analysing nucleic acid from the sample for SNPs in the 16S rRNA gene (or 16S rRNA or DNA copy thereof) at positions corresponding to any one of positions 273, 378, 412, 440, 488, 647 and 653 of the 16S rRNA gene set forth in SEQ ID NO:1, wherein: a T at position 412 indicates that the sample includes the bacterium Staphylococcus aureus and/or S. epidermidis; a G at position 647 indicates that the sample includes the bacterium Enterococcusfaecalisand/or Enterococcus faecium; and a C at positions 440 and 647 and a T at position 488 indicates that the sample includes the bacterium Serratia marcescens and/or Enterobacteraerogenes.
[00230] Further to the above, methods of the present invention can also be used to identify each of Staphylococcus aureus; S. epidermidis; Enterococcusfaecalis; Enterococcusfaecium; Serratia marcescens; and Enterobacter aerogenes in a sample. The methods include further analysing the nucleic acid from the sample with high-resolution melt analysis (see 3.8, below). High-resolution melt analysis allows nucleic acid sequences from different bacterium but containing the same SNP(s) to be differentiated from one another based upon variations in the surrounding nucleotide bases.
[00231] In another embodiment, the present invention provides methods for identifying bacterium selected from among: Escherichia coli, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Proteus mirabilis, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Enterococcusfaecalis, Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens, Corynebacterium jeikeium, Bacteroides fragilis, Neisseria meningitidis, Haemophilus influenzae, Serratia marcescens, Salmonella sp., and Staphylococcus epidermidis. The general rules for identifying the above bacterial species within a sample using the above SNPs are depicted in Table 2.
[00232] Similarly, as demonstrated herein, polymorphisms at nucleotide positions of the gene encoding 16S rRNA (and thus of the rRNA molecule itself) that correspond to positions 746, 764, 771, or 785 of the Bacillus anthracis 16S rRNA gene as set forth in SEQ ID NO:43 (or positions 737, 755, 762, or 776 of the 16S rRNA gene as set forth in SEQ ID NO:1) can be used to identify bacterium within a sample, particularly including mammalian (e.g., human) pathogens (including pathogens known as Security Sensitive Biological Agents).
[00233] In one embodiment, there is provided methods for identifying bacterium selected from among: Bacillus anthracis, Clostridiumbotulinum type A, Clostridium botulinum type B, Clostridium botulinum type C, Clostridium botulinum type D, Clostridium botulinum type G,
Yersinia pestis, Francisellatularensis, Vibrio cholerae and Burkholderiapseudomallei.
[00234] The general rules for identifying the above bacterial species within a sample using the above SNPs are depicted in Table 3 and/or Table 6.
[00235] From the above bacteria, the bacterium Bacillus anthracis can be identified in a sample by analysing nucleic acid from the sample for an SNP in the 16S rRNA gene (or 16S rRNA or DNA copy thereof) of SEQ ID NO. 43, where a T at position 746, A at position 764, C at position 771 and G at position 785 indicates that the bacterium is Bacillus anthracis.
[00236] From the above bacteria, bacterium selected from Clostridium botulinum type A or Clostridium botulinum type B can be identified in a sample by analysing nucleic acid from the sample for an SNP in the 16S rRNA gene (or 16S rRNA or DNA copy thereof) of SEQ ID NO. 43, where a T at position 746, G at position 764, C at position 771 and T at position 785 indicates that the bacterium is Clostridium botulinum type A or Clostridiumbotulinum type B.
[00237] From the above bacteria, the bacterium Clostridium botulinum type C can be identified in a sample by analysing nucleic acid from the sample for an SNP in the 16S rRNA gene (or 16S rRNA or DNA copy thereof) of SEQ ID NO. 43, where a T at position 746, A at position 764, T at position 771 and T at position 785 indicates that the bacterium is Clostridium botulinum type C.
[00238] From the above bacteria, the bacterium Clostridium botulinum type D can be identified in a sample by analysing nucleic acid from the sample for an SNP in the 16S rRNA gene (or 16S rRNA or DNA copy thereof) of SEQ ID NO. 43, where a C at position 746, A at position 764, T at position 771 and T at position 785 indicates that the bacterium is Clostridium botulinum type D.
[00239] From the above bacteria, the bacterium Clostridium botulinum type G can be identified in a sample by analysing nucleic acid from the sample for an SNP in the 16S rRNA gene (or 16S rRNA or DNA copy thereof) of SEQ ID NO. 43, where a T at position 746, G at position 764, C at position 771 and G at position 785 indicates that the bacterium is Clostridium botulinum type G.
[00240] From the above bacteria, the bacterium Yersinia pestis can be identified in a sample by analysing nucleic acid from the sample for an SNP in the 16S rRNA gene (or 16S rRNA or DNA copy thereof) of SEQ ID NO. 43, where a C at position 746, G at position 764, T at position 771 and G at position 785 indicates that the bacterium is Yersinia pestis.
[00241] From the above bacteria, the bacterium Francisellatularensis can be identified in a sample by analysing nucleic acid from the sample for an SNP in the 16S rRNA gene (or 16S rRNA or DNA copy thereof) of SEQ ID NO. 43, where a T at position 746, A at position 764, G at position 771 and G at position 785 indicates that the bacterium is Francisellatularensis.
[00242] From the above bacteria, the bacterium Vibrio cholerae can be identified in a sample by analysing nucleic acid from the sample for an SNP in the 16S rRNA gene (or 16S rRNA or DNA copy thereof) of SEQ ID NO. 43, where a C at position 746, A at position 764, T at position 771 and G at position 785indicates that the bacterium is Vibrio cholerae.
[00243] From the above bacteria, the bacterium Burkholderiapseudomallei can be identified in a sample by analysing nucleic acid from the sample for an SNP in the 16S rRNA gene (or 16S rRNA or DNA copy thereof) of SEQ ID NO. 43, where a C at position 746, G at position 764, C at position 771 and G at position 785 indicates that the bacterium is Burkholderia pseudomallei.
[00244] Further to the above, methods of the present invention may also be used to identify each of Bacillus anthracis, Clostridium botulinum type A, Clostridium botulinum type B, Clostridium botulinum type C, Clostridium botulinum type D, Clostridium botulinum type G, Yersinia pestis, Francisella tularensis, Vibrio cholerae and Burkholderia pseudomallei in a sample. The methods include further analysing the nucleic acid from the sample with high resolution melt analysis (see 3.8, below).
[00245] Similarly, as demonstrated herein, polymorphisms at nucleotide positions of the gene encoding 18S rRNA (and thus of the rRNA molecule itself) that correspond to positions 343, 371, 388, 416, and 467 of the Candida albicans 18S rRNA gene set forth in SEQ ID NO: 37 can be used to identify bacterium within a sample, particularly including mammalian (e.g., human) pathogens.
[00246] In one embodiment, there is provided methods for identifying a yeast organism or filamentous fungi selected from among: Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, Fusarium spp., Aspergillus fumigatus, and Cryptococcus neoformans.
[00247] The general rules for identifying the above bacterial species within a sample using the above SNPs are depicted in Table 4 and/or Table 7.
[00248] From the above yeast organism or filamentous fungi, the yeast organism Candida albicans can be identified in a sample by analysing nucleic acid from the sample for an SNP in the 18S rRNA gene (or 18S rRNA or DNA copy thereof) of SEQ ID NO. 37, where a C at position 343, A at position 371, T at position 388, G at position 416 and G at position 467 indicates that the yeast organism is Candida albicans.
[00249] From the above yeast organism or filamentous fungi, the yeast organism Candida tropicaliscan be identified in a sample by analysing nucleic acid from the sample for an SNP in the 18S rRNA gene (or 18S rRNA or DNA copy thereof) of SEQ ID NO. 37, where a C at position 343, A at position 371, C at position 388, G at position 416 and C at position 467 indicates that the yeast organism is Candida tropicalis.
[00250] From the above yeast organism or filamentous fungi, the yeast organism Candida parapsilosiscan be identified in a sample by analysing nucleic acid from the sample for an SNP in the 18S rRNA gene (or 18S rRNA or DNA copy thereof) of SEQ ID NO. 37, where a C at position 343, A at position 371, C at position 388, G at position 416 and G at position 467 indicates that the yeast organism is Candidaparapsilosis.
[00251] From the above yeast organism or filamentous fungi, the yeast organism Candida glabratacan be identified in a sample by analysing nucleic acid from the sample for an SNP in the 18S rRNA gene (or 18S rRNA or DNA copy thereof) of SEQ ID NO. 37, where a T at position 343, A at position 371, C at position 388, G at position 416 and G at position 467 indicates that the yeast organism is Candidaglabrata.
[00252] From the above yeast organism or filamentous fungi, the yeast organism Cryptococcus neoformans can be identified in a sample by analysing nucleic acid from the sample for an SNP in the 18S rRNA gene (or 18S rRNA or DNA copy thereof) of SEQ ID NO. 37, where a C at position 343, A at position 371, T at position 388, T at position 416 and G at position 467 indicates that the yeast organism is Cryptococcus neoformans.
[00253] From the above yeast organism or filamentous fungi, the filamentous fungi Fusarium spp. can be identified in a sample by analysing nucleic acid from the sample for an SNP in the 18S rRNA gene (or 18S rRNA or DNA copy thereof) of SEQ ID NO. 37, where a C at position 343, C at position 371, T at position 388, T at position 416 and G at position 467 indicates that the filamentous fungi is Fusariumspp.
[00254] From the above yeast organism or filamentous fungi, the filamentous fungi Aspergillus fumigatus can be identified in a sample by analysing nucleic acid from the sample for an SNP in the 18S rRNA gene (or 18S rRNA or DNA copy thereof) of SEQ ID NO. 37, where a C at position 343, C at position 371, T at position 388, C at position 416 and G at position 467 indicates that the filamentous fungi is Aspergillusfumigatus.
[00255] Further to the above, methods of the present invention may also be used to identify each of Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, Fusariumspp., Aspergillusfumigatus, and Cryptococcus neoformans in a sample. The methods include further analysing the nucleic acid from the sample with high-resolution melt analysis (see 3.8, below).
3. Screening for specific polymorphisms to identify and/or classify bacteria, yeast organisms andfilamentousfungi according to the invention
[00256] Steps/techniques of isolating a biological sample from a subject, processing a biological sample, genomic DNA extraction, RNA extraction, DNA detection and characterisation, RNA detection and characterisation, DNA sequencing, DNA sequence analyses, SNP genotyping studies, RNA location and identification, RNA profiling and RNA screening, RNA sequencing, RNA sequence analyses can be carried out in any suitable way.
[00257] Any method known in the art to detect one or more SNPs can be used in the methods described herein to classify and/or identify bacterial species within a sample. In particular embodiments, the methods also facilitate in the narrowing down or, in some cases, confirming of one bacterial species (or yeast organism or filamentous fungi species) over another. Numerous methods are known in the art for determining the nucleotide occurrence at a particular position corresponding to a single nucleotide polymorphism in a sample. The various tools for the detection of polymorphisms include, but are not limited to, DNA sequencing, scanning techniques, hybridization based techniques, extension based analysis, high-resolution melting analysis, incorporation based techniques, restriction enzyme based analysis and ligation based techniques.
[00258] The methods according to the present invention can identify polymorphisms described herein within the 16S rRNA or 18S rRNA genes, within the 16S rRNA or 18S rRNA molecule or within DNA copies thereof, and for either strand. In some examples, the methods of detecting the polymorphisms utilise a first step of amplification, and amplification can be from the 16S rRNA or 18S rRNA gene or from DNA copies of the 16S rRNA or 18S rRNA molecule.
[00259] The nucleic acid may be from a biological sample from a subject or from an environmental sample, such as an air, soil or water sample, a filtrate, a food or manufactured product, or swap from a surface, such as from a medical instrument or work place surface. The subject may be a human subject or non-human subject, such as a mammalian subject, such as a primates, livestock animals (e.g., sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs) and captive wild animals (e.g., koalas, bears, wild cats, wild dogs, wolves, dingoes, foxes and the like). Biological samples from a subject may be from any part of the subject's body, including but not limited to bodily fluids such as blood, saliva, sputum, urine, cerebrospinal fluid, faeces, cells, tissue or biopsies. In other examples, the nucleic acid is obtained from cultured cells.
[00260] The nucleic acid that is analysed according to the methods of the present invention may be analysed while within the sample, or may first be extracted from the sample, e.g., isolated from the sample prior to analysis. Any method for isolating nucleic acid from a sample can be used in the methods of the present invention, and such methods are well known to those of skill in the art. The extracted nucleic acid can include DNA and/or RNA (including mRNA or rRNA). In some examples, a further step of reverse transcription can be included in the methods prior to analysis. Thus, the nucleic acid to be analysed can include the 16S rRNA gene, 18S rRNA gene, 16S rRNA, 18S rRNA, a DNA copy of the 16S rRNA or the 18S rRNA or any combination thereof. The nucleic acid can also contain portions of the 16S rRNA gene, 18S rRNA gene, 16S rRNA, 18S rRNA or a DNA copy of the 16S rRNA or the 18S rRNA, providing the portions containing the nucleic acid positions that are being analysed for SNPs.
[00261] In some instances, the methods include amplification of the nucleic acid. In such instances, suitable nucleic acid amplification techniques are well known to a person or ordinary skill in the art, and include polymerase chain reaction (PCR) as for example described in Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, Inc. 1994-1998, strand displacement amplification (SDA) as for example described in US Patent No. 5,422,252, rolling circle replication (RCR) as for example described in Liu et al. (1996) and in WO 92/01813 and WO 97/19193, nucleic acid sequence-based amplification (NASBA) as for example described in Sooknanan et al., (1994), ligase chain reaction (LCR), simple sequence repeat analysis (SSR), branched DNA amplification assay (b-DNA), transcription amplification and self-sustained sequence replication, and Q-0 replicase amplification as for example described in Tyagi et al. (1996).
[00262] Such methods can utilise one or more oligonucleotide probes or primers, including, for example, an amplification primer pair, that selectively hybridize to a target polynucleotide, which contains one or more SNPs. Oligonucleotide probes useful in practicing a method of the invention can include, for example, an oligonucleotide that is complementary to and spans a portion of the target polynucleotide, including the position of the SNP, which the presence of a specific nucleotide at the polymorphic site (i.e., the SNP) is detected by the presence or absence of selective hybridization of the probe. Such a method can further include contacting the target polynucleotide and hybridized oligonucleotide with an endonuclease, and detecting the presence or absence of a cleavage product of the probe, depending on whether the nucleotide occurrence at the polymorphic site is complementary to the corresponding nucleotide of the probe.
[00263] Primers may be manufactured using any convenient method of synthesis. Examples of such methods may be found in "Protocols for Oligonucleotides and Analogues; Synthesis and Properties", Methods in Molecular Biology Series, Volume 20, Ed. Sudhir Agrawal, Humana ISBN: 0-89603-247-7, 1993. The primers may also be labelled to facilitate detection.
[00264] Any method useful for the detection of SNPs can be used in the present invention, and many different methods are known in the art for SNP genotyping (for review see Syvanen, A. C. (2001); Kim, S. and Misra, A., (2007)). Such methodology may consist of the use of three steps in succession, including a "reaction" (e.g., hybridization, ligation, extension and cleavage) followed by "separation" (e.g., solid phase microtitre plates, microparticles or arrays, gel electrophoresis, solution-phase homogenous or semi-homogenous). No single ideal SNP genotyping method exists for all applications, and it is well within the skill of a skilled artisan to determine the most appropriate method given the various parameters, such as sample size and number of SNPs to be analysed.
[00265] Example technologies that particularly lend themselves to clinical use and that rely on querying small numbers of SNPs, are fast, sensitive (through amplification of nucleic acid in the sample), one-step, output measured in real-time, able to be multiplexed and automated, comparatively inexpensive, and accurate include, but are not limited to, TaqMan@ assays (5' nuclease assay, Applied Biosystems), high-resolution melt analysis, molecular beacon probes such as LUX@ (Invitrogen) or Scorpion@ probes (Sigma Aldrich), and Template Directed Dye Incorporation (TDI, Perkin Elmer).
[00266] For example, TaqMan@ (Applied Biosystems) uses a combination of hybridization with allele-specific probes, solution phase homogenous, and fluorescence resonance energy transfer. The TaqMan@ assay relies on forward and reverse primers and Taq DNA polymerase to amplify nucleic acid in conjunction with 5'-nuclease activity of Taq DNA polymerase to degrade a labelled probe designed to bind across the SNP site(s). Reaction, separation and detection can all be performed at the same time and results read in real-time as the reaction proceeds. While such an approach does not lend itself to analysing large numbers of SNPs simultaneously it is particularly suitable for querying small numbers of SNPs quickly, sensitively and accurately at a reasonable cost.
[00267] Although some methods may be more suitable than others, any method known in the art to detect one or more SNPs can be used in the methods described herein to classify and/or identify bacteria and/or bacterium in a sample. Non-limiting examples of such methods are described below.
3.1 Nucleic acid sequencing techniques
[00268] In some embodiments, the polymorphism is identified through nucleic acid sequencing techniques. Specifically, amplification products which span a SNP locus can be sequenced using traditional sequencing methodologies (e.g., the "dideoxy-mediated chain termination method", also known as the "Sanger Method" (Sanger, F., et al. (1975)) and the "chemical degradation method", also known as the "Maxam-Gilbert method" (Maxam, A. M., et al., 1977) both references are herein incorporated by reference to determine the nucleotide occurrence at the SNP loci.
[00269] Boyce-Jacino et al., US Patent No. 6,294,336 provides a solid phase sequencing method for determining the sequence of nucleic acid molecules (either DNA or RNA) by utilizing a primer that selectively binds a polynucleotide target at a site wherein the SNP is the most 3' nucleotide selectively bound to the target. Other sequencing technologies such as a Denaturing High Pressure Liquid Chromatography or mass spectrometry may also be employed.
[00270] In other illustrative examples, the sequencing method comprises a technique known as PyrosequencingTM. The approach is based on the generation of pyrophosphate whenever a deoxynucleotides is incorporated during polymerization of DNA. The generation of pyrophosphate is coupled to a luciferase-catalysed reaction resulting in light emission if the particular deoxynucleotides added is incorporated, yielding a quantitative and distinctive pyrogram. Sample processing includes PCR amplification with a biotinylated primer, isolation of the biotinylated single strand amplicon on streptavidin coated beads (or other solid phase) and annealing of a sequencing primer. Samples are then analysed by a Pyrosequence TM, which adds a number of enzymes and substrates required for the indicator reaction, including sulfurylase and luciferase, as well as pyrase for degradation of unincorporated molecules. The sample is then interrogated by addition of the four deoxynucleotides. Light emission can be detected by a charge coupled device (CCD) camera and is proportional to the number of nucleotides incorporated. Results are automatically assigned by pattern recognition.
[00271] Alternatively, methods of the invention can identify nucleotide occurrences at polymorphic sites within a nucleic acid using a "micro-sequencing" method. Micro-sequencing methods determine the identity of only a single nucleotide at a "predetermined" site. Such methods have particular utility in determining the presence and identity of polymorphisms in a target polynucleotide. Such micro-sequencing methods, as well as other methods for determining the nucleotide occurrence at a polymorphic site are discussed in US Patent No. 6,294,336, which is incorporated herein by reference.
[00272] Micro-sequencing methods include the Genetic Bit AnalysisTM method disclosed in WO 92/15712. Additional, primer-guided, nucleotide incorporation procedures for assaying polymorphic sites in DNA have also been described (Komher, J.S., et al., 1989); Sokolov, B.P., 1990; Syvanen, A.C., et al., 1990; Kuppuswamy, M.N., et al., 1991; Prezant, T.R., et al. 1992; Ugozzoli, L., et al., 1992; Nyren, P., et al. 1993; and WO 89/10414). These methods differ from Genetic Bit AnalysisTM in that they all rely on incorporation of labelled deoxynucleotides to discriminate between bases at a polymorphic site. In such a format, since the signal is proportional to the number of deoxynucleotides incorporated, polymorphisms that occur in runs of the same nucleotide can result in signals that are proportional to the length of the run (Syvanen, A.C., et al. 1993).
[00273] Further micro-sequencing methods have been provided by US Patent No. 4,656,127 and French Patent No. 2,650,840 (WO 91/02087) which involve a solution-based method for determining the identity of a nucleotide of a polymorphic site. As in the method of the US Patent, a primer is employed that is complementary to allelic sequences immediately 3' to a polymorphic site.
[00274] In other illustrative examples, US Patent No. 5,002,867, for example, describes a method for determining nucleic acid sequences via hybridization with multiple mixtures of oligonucleotide probes. In accordance with such methods, the sequence of a target polynucleotide is determined by permitting the target to sequentially hybridize with sets of probes having an invariant nucleotide at one position, and variant nucleotides at other positions. The method determines the nucleotide sequence of the target by hybridizing the target with a set of probes, and then determining the number of sites that at least one member of the set is capable of hybridizing to the target (i.e., the number of matches). The procedure is typically repeated until each member of a set of probes has been tested.
[00275] Alternatively, the template-directed dye-terminator incorporation assay with fluorescence polarization detection (FP-TDI) assay (Chen et al. 1999) is a version of the primer extension assay that is also called mini-sequencing or the single base extension assay (Syvanen, A.C., et al., 1990). The primer extension assay is capable of detecting SNPs. The DNA sequencing protocol ascertains the nature of the one base immediately 3' to the SNP-specific sequencing primer that is annealed to the target DNA immediately upstream from the polymorphic site. In the presence of DNA polymerase and the appropriate dideoxyribonucleoside triphosphate (ddNTP), the primer is extended specifically by one base as dictated by the target DNA sequence at the polymorphic site. By determining which ddNTP is incorporated, the allele(s) present in the target DNA can be inferred.
3.2 Polymorphism hybridization based techniques
[00276] Hybridization techniques for detecting polymorphisms within a nucleotide sequence can include, but are not restricted to the TaqMan@ assay (Applied Biosystems), dot blots, reverse dot blot, Multiplex-allele-specific diagnostic assays (MASDA), Dynamic allele-specific hybridization (DASH) (Jobs et al. 2003), molecular beacons and Southern Blots.
[00277] The TaqMan@ assay (also known as a 5' nuclease assay or 5' digestion assay) for identifying SNPs within a nucleotide sequence is based on the nuclease activity of Taq polymerase that displaces and cleaves the oligonucleotide probe hybridized to the target DNA, generating a fluorescent signal. TaqMan@ probes specific for a particular SNP are required, with each probe having different fluorescent dyes attached to the 5' end and quencher attached to the 3' end. When the probes are intact, the quencher interacts with the fluorophore by fluorescence resonance energy transfer (FRET), quenching their fluorescence. During the PCT annealing step, the TaqMan@ probes hybridize to the target DNA. In the extension step, the fluorescent dye is cleaved by the nuclease activity of the Taq polymerase, leading to an increase in fluorescence by the reporter dye. Mismatch probes are displaced without fragmentation. The genotype of a sample is determined by measuring the signal intensity of the two different dyes.
[00278] Another useful SNP identification method includes DASH (dynamic allele-specific hybridization), which encompasses dynamic tracking of probe (oligonucleotide) to target (PCR product) hybridization as the reaction temperature is steadily increased to identify polymorphisms (Prince, J.A., et al. 2001).
[00279] In some embodiments, multiplex-allele-specific diagnostic assays (MASDA) can be used for the analysis of a large number of samples (>500). MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotides (ASO) probes. Any probes complementary to specific polymorphisms present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s).
[00280] There are several alternative hybridization-based techniques, including, among others, molecular beacons, and Scorpion@ probes (Tyagi, S. and Kramer, F.R. 1996; Thelwell et al., 2000). Molecular beacons are comprised of oligonucleotides that have fluorescent reporter and quencher dyes at their 5' and 3' ends. The central portion of the oligonucleotide hybridizes across the target sequence, but the 5' and 3' flanking regions are complementary to each other. When not hybridized to their target sequence, the 5' and 3' flanking regions hybridize to form a stem-loop structure, and there is little fluorescence because of the proximity of the reporter and quencher dyes. However, upon hybridization to their target sequence, the dyes are separated and there is a large increase in fluorescence. Mismatched probe-target hybrids dissociate at substantially lower temperature than exactly complementary hybrids. There are a number of variations of the "beacon" approach. Scorpion@ probes are similar but incorporate a PCR primer sequence as part of the probe. A more recent "duplex" format has also been developed.
[00281] In some embodiments, a further method of identifying a SNP comprises the SNP ITTM method (Orchid BioSciences, Inc., Princeton, N.J.). In general, SNP-ITTM is a 3-step primer extension reaction. In the first step a target polynucleotide is isolated from a sample by hybridization to a capture primer, which provides a first level of specificity. In a second step, the capture primer is extended from a terminating nucleotide triphosphate at the target SNP site, which provides a second level of specificity. In a third step, the extended nucleotide triphosphate can be detected using a variety of known formats, including: direct fluorescence, indirect fluorescence, an indirect colorimetric assay, mass spectrometry, fluorescence polarization, etc. Reactions can be processed in 384-well format in an automated format using a SNPstreamTMinstrument (Orchid BioSciences, Inc., Princeton, N.J.).
[00282] In these embodiments, the amplification products can be detected by Southern Blot analysis with or without using radioactive probes. In one such method, for example, a small sample of DNA containing a very low level of the nucleic acid sequence of the polymorphic locus is amplified, and analyzed via a Southern Blotting technique or similarly, using a dot blot analysis. The use of non-radioactive probes or labels is facilitated by the high level of the amplified signal. Alternatively, probes used to detect the amplified products can be directly or indirectly detectably labelled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme.
[00283] Hybridization conditions, such as salt concentration and temperature can also be adjusted for the nucleotide sequence to be screened. Southern blotting and hybridization protocols are described in Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley-Interscience), pages 2.9.1-2.9.10. Probes can be labelled for hybridization with random oligomers and the Klenow fragment of DNA polymerase. Very high specific activity probes can be obtained using commercially available kits such as the Ready To-Go DNA Labeling Beads (Pharmacia Biotech), following the manufacturer's protocol. Possible competition probes having high repeat sequence content, and stringency of hybridization and wash down will be determined individually for each probe used. Alternatively, fragments of a candidate sequence may be generated by PCR, the specificity may be verified using a rodent-human somatic cell hybrid panel, and sub-cloning the fragment. This allows for a large prep for sequencing and use as a probe. Once a given gene fragment has been characterized, small probe preparations can be achieved by gel or column purifying the PCR product.
[00284] Suitable materials that cane be used in the dot blot, reverse dot blot, multiplex and MASDA formats are well known in the art and include, but are not limited to nylon and nitrocellulose membranes.
3.3 Polymorphism scanning techniques
[00285] Scanning techniques contemplated by the present invention for detecting polymorphisms within a nucleotide sequence can include, but are not restricted to, chemical mismatch cleavage (CMC) (Saleeba, J. A et al., 1992), mismatch repair enzymes cleavage (MREC) (Lu, A.L. and Hsu, I.C, 1992), chemical cleavage techniques, denaturing gradient gel electrophoresis (DGGE) (Wartell et al., 1990; Sheffield et al., 1989), temperature gradient gel electrophoresis (TGGE) (Salimullah, et al. 2005), constant denaturant gel electrophoresis (CDGE), single strand conformation polymorphism (SSCP) analysis (Kumar, D. et al., 2006), heteroduplex analysis (HA) (Nagamine, C.M., et al., 1989), microsatellite marker analysis and single strand polymorphism assays (SSPA).
[00286] In some embodiments, the SNPs of the present invention are detected through CMC, wherein a radiolabeled DNA wild type sequence (i.e., probe) is hybridized to an amplified sequence containing the putative alteration to form a heteroduplex. A chemical modification, followed by piperidine cleavage, is used to remove the mismatch bubble in the heteroduplex. Gel electrophoresis of the denatured heteroduplex and autoradiography allow visualisation of the cleavage product. Osmium tetroxide is used for the modification of mispaired thymidines and hydroxylamine for mismatched cytosines. Additionally, labeling the antisense strand of the probe DNA allows the detection of adenosine and guanosine mismatches. The chemical cleavage of mismatch can be used to detect almost 100% of mutations in long DNA fragments. Moreover, this method provides the precise characterization and the exact location of the mutation within the tested fragment. Recently, the method has been amended to make CMC more suitable for automation by using fluorescent primers also enabling multiplexing and thereby reducing the number of manipulations. Alternatively, fluorescently labeled dUTPs incorporated via PCR allow the internal labelling of both target and probe DNA strands and therefore labelling of each possible hybrid, doubling the chances of mutation detection and virtually guaranteeing 100% detection.
[00287] In other embodiments, the mismatch repair enzymes cleavage (MREC) assay is used to identify SNPs of the present invention. MREC relies on nicking enzyme systems specific for mismatch-containing DNA. The sequence of interest is amplified by PCR and homo- and heteroduplex species may be generated at the end of the PCR, by denaturing and allowing to re-anneal the amplified products. These hybrids are treated with mismatch repair enzymes and then analysed by denaturing gel electrophoresis. The MREC assay makes use of three mismatch repair enzymes. The MutY endonuclease removes adenines from the mismatches and is useful to detect both A/T and C/G transversions and G/C and T/A transitions. Mammalian thymine glycosylase removes thymines from T/G, T/C and T/T mismatches and is useful to detect G/C and A/T transitions as well as A/T and G/C and T/A and A/T transversions. The all-type endonuclease or topoisomerase I from human or calf thymus can recognize all eight mismatches and can be used to scan any nucleotide substitution. MREC can use specific labels which can be incorporated into both DNA strands, thus allowing all four possible nucleotide substitutions in a given site to be identified.
[00288] In some embodiments, chemical cleavage analysis as described in US Patent No. 5,217,863 is used for identifying SNPs within nucleotide sequences. Like heteroduplex analysis, chemical cleavage detects different properties that result when mismatched allelic sequences hybridize with each other. Instead of detecting this difference as an altered migration rate on a gel, the difference is detected in altered susceptibility of the hybrid to chemical cleavage using, for example, hydroxylamine, or osmium tetroxide, followed by piperidine.
[00289] Among the cleavage methods contemplated by the present invention, RNAse A relies on the principle of heteroduplex mismatch analysis. In the RNAse A cleavage method, RNA-DNA heteroduplex between radiolabeled riboprobe and a DNA, obtained by PCR amplification, is enzymatically cleaved by RNAse A, by exploiting the ability of RNAse A to cleave single-stranded RNA at the points of mismatches in RNA:DNA hybrids. This is followed by electrophoresis and autoradiography. The presence and location of a mutation are indicated by a cleavage product of a given size (Meyers, R.M., et al., 1985; Gibbs, R.A. and Caskey, T., 1987).
[00290] DNA probes also can be used to detect mismatches, through enzymatic or chemical cleavage; see, e.g., Cotton, et al., 1988; Shenk et al., 1975; and Novack et al., 1986.
[00291] In some embodiments, the Invader@ assay (Third Wave TM Technology) may be employed to scan for polymorphisms within the 16S rRNA genes of the present invention. For example, the Invader@ assay is based on the specificity of recognition, and cleavage, by a Flap endonuclease, of the three dimensional structure formed when two overlapping oligonucleotides hybridize perfectly to a target DNA (Lyamichev, V. et al., 1999).
[00292] Alternatively, denaturing gradient gel electrophoresis (DGGE) is a useful technique to separate and identify sequence variants. DGGE is typically performed in constant concentration polyacrylamide gel slabs, cast in the presence of linearly increasing amounts of a denaturing agent (usually formamide and urea, cathode to anode). A variant of DGGE employs temperature gradients along the migration path and is known as TGGE. Separation by DGGE or TGGE is based on the fact that the electrophoretic mobility in a gel of a partially melted DNA molecule is greatly reduced as compared to an unmelted molecule.
[00293] In some embodiments, constant denaturant gel electrophoresis (CDGE) is useful for detecting SNPs within a nucleotide sequence, as described in detail in Smith-Sorenson et al., 1993. A given DNA duplex melts in a predetermined, characteristic fashion in a gel of a constant denaturant. Mutations alter this movement. An abnormally migrating fragment is isolated and sequenced to determine the specific mutation.
[00294] In other embodiments, single-strand conformation polymorphism (SSCP) analysis provides a method for detecting SNPs of the present invention. SSCP is a method based on a change in mobility of separated single-strand DNA molecules in non-denaturing polyacrylamide gel electrophoresis. Electrophoretic mobility depends on both size and shape of a molecule, and single-stranded DNA molecules fold back on themselves and generate secondary structures, which are determined by intra-molecular interactions in a sequence dependent manner. A single nucleotide substitution can alter the secondary structure and, consequently, the electrophoretic mobility of the single strands, resulting in band shifts on autoradiographs. The ability of a given nucleotide variation to alter the conformation of the single strands is not predictable on the basis of an adequate theoretical model and base changes occurring in a loop or in a long stable stem of the secondary structure might not be detected by SSCP. Standard SSCP reaches maximal reliability in detecting sequence alterations in fragments of 150-200 bp. More advanced protocols, allowing the detection of mutations at sensitivity equal to that of the radioactively-based SSCP analysis, have been developed. These methods use fluorescence-labeled primers in the PCR and analyze the products with a fluorescence-based automated sequencing machine. Multi-colour fluorescent SSCP also allows including an internal standard in every lane, which can be used to compare data from each lane with respect to each other. Other variants to increase the detection rate include a dideoxy sequencing approach based on dideoxy fingerprinting (ddF) and restriction endonuclease fingerprinting (REF).
[00295] The ddF method is a combination of SSCP and Sanger dideoxy sequencing, which involves non-denaturing gel electrophoresis of a Sanger sequencing reaction with one dideoxynucleotide. In this way, for example, a 250-bp fragment can be screened to identify a SNP. REF is a more complex modification of SSCP allowing the screening of more than 1 kb fragments. For REF, a target sequence is amplified with PCR, digested independently with five to six different restriction endonucleases and analyzed by SSCP on a non-denaturing gel. In the case of six restriction enzymes being used, a sequence variation will be present in six different restriction fragments, thus generating 12 different single-stranded segments. A mobility shift in any one of these fragments is sufficient to pinpoint the presence of a SNP of the invention. The restriction pattern obtained enables localization of an alteration in the region examined.
[00296] In some embodiments, heteroduplex analysis (HA) detects single base substitutions in PCR products or nucleotide sequences. HA can be rapidly performed without radioisotopes or specialized equipment. The HA method takes advantage of the formation of heteroduplexes between sequences with differing nucleotides at one or more positions by heating and renaturing of PCR products. Due to a more open double-stranded configuration surrounding the mismatched bases, heteroduplexes migrate slower than their corresponding homoduplexes, and are then detected as bands of reduced mobility. The ability of a particular single base substitution to be detected by the HA method cannot be predicted merely by knowing the mismatched bases since the adjacent nucleotides have a substantial effect on the configuration of the mismatched region and length-based separation will clearly miss nucleotide substitutions. Optimization of the temperature, gel cross-linking and concentration of acrylamide used as well as glycerol and sucrose enhance the resolution of mutated samples. The HA method can be rapidly performed without radioisotopes or specialized equipment and screens large numbers of samples for known mutations and polymorphisms in sequenced genes. When HA is used in combination with SSCP, up to 100% of all alterations in a DNA fragment can be easily detected.
[00297] In some embodiments, the use of proteins that recognize nucleotide mismatches, such as the E. coli mutS protein can be used to detect a polymorphism within 16S rRNA of the present invention (Modrich 1991). In the mutS assay, the protein binds only to sequences that contain a nucleotide mismatch in a heteroduplex between two sequences.
[00298] In further embodiments, polymorphism detection can be performed using microsatellite marker analysis. Microsatellite markers with average genome spacing, for example of about 10 centimorgans (cM), can be employed using standard DNA isolation methods known in the art.
[00299] SSPA analysis and the closely related heteroduplex analysis methods described above may be used for screening for single -base polymorphisms (Orita, M. et al., 1989).
3.4 Nucleotide Arrays and Gene Chipsfor polymorphism analysis
[00300] The invention further contemplates methods of identifying SNPs through the use of an array of oligonucleotides, wherein discrete positions on the array are complementary to one or more of the sequences containing the SNPs of the present invention, e.g. oligonucleotides of at least 12 nt, at least about 15 nt, at least about 18 nt, at least about 20 nt, or at least about 25 nt, or longer, and including the sequence flanking the polymorphic position. Such an array may comprise a series of oligonucleotides, each of which can specifically hybridize to a different polymorphism. For examples of arrays, see Hacia et al., 1996 and De Risi et al., 1996.
[00301] A nucleotide array can include all or a subset of the polymorphisms of the invention, as required. One or more polymorphic forms may be present in the array. The oligonucleotide sequence on the array is generally at least about 12 nt in length, at least about 15 nt, at least about 18 nt, at least about 20 nt, or at least about 25 nt, or more, such as 100 to 200 nt in length. For examples of arrays, see Ramsay 1998; Hacia et al., 1996; Lockhart et al., 1996; and De Risi et al., 1996.
[00302] A number of methods are available for creating micro-arrays of biological samples, such as arrays of DNA samples to be used in DNA hybridization assays. Examples of such arrays are discussed in detail in PCT Application No. W095/35505; U.S. Patent Application number. 5,445,934; and Drmanac et al., 1993. Yershov et al. 1996 describes an alternative construction of an oligonucleotide array. The construction and use of oligonucleotide arrays are reviewed by Ramsay (1998).
[00303] Methods of using high-density oligonucleotide arrays for identifying polymorphisms within nucleotide sequences are known in the art. For example, Milosavljevic et al., 1996 describe DNA sequence recognition by hybridization to short oligomers (see also, Drmanac et al., 1998; and Drmanac and Drmanac, 1999). The use of arrays for identification of unknown mutations is proposed by Ginot 1997.
[00304] Detection of known mutations is described in Hacia et al. 1996; Cronin et al., 1996; and others. The use of arrays in genetic mapping is discussed in Chee et al., 1996; Sapolsky and Lishutz, 1996; and Shoemaker et al., 1996.
[00305] Quantitative monitoring of gene expression patterns with a complementary DNA microarray is described in Schena et al., 1995; and DeRisi et al., 1997. Wodicka et al., 1997 performs genome wide expression monitoring in S. cerevisiae.
[00306] High-density microarrays of oligonucleotides are known in the art and are commercially available. The sequence of oligonucleotides on the array will correspond to a known target sequences. The length of oligonucleotide present on the array is an important factor in how sensitive hybridization will be to the presence of a mismatch. Usually oligonucleotides will be at least about 12 nt in length, more usually at least about 15 nt in length, preferably at least about 20 nt in length and more preferably at least about 25 nt in length, and will be not longer than about 35 nt in length, usually not more than about 30 nt in length.
[00307] Methods of producing large arrays of oligonucleotides are described in US Patent No. 5,134,854 and US Patent No. 5,445,934 using light-directed synthesis techniques. Using a computer-controlled system, a heterogeneous array of monomers is converted, through simultaneous coupling at a number of reaction sites, into a heterogeneous array of polymers. Alternatively, microarrays are generated by deposition of pre-synthesized oligonucleotides onto a solid substrate, for example as described in International Publication WO 95/35505.
[00308] Microarrays can be scanned to detect hybridization of the labeled genome samples. Methods and devices for detecting fluorescently marked targets on devices are known in the art. Generally such detection devices include a microscope and light source for directing light at a substrate. A photon counter detects fluorescence from the substrate, while an x-y translation stage varies the location of the substrate. A confocal detection device that may be used in the subject methods is described in US Patent No. 5,631,734. A scanning laser microscope is described in Shalon et al. 1996. A scan, using the appropriate excitation line, is performed for each fluorophore used. The digital images generated from the scan are then combined for subsequent analysis. For any particular array element, the ratio of the fluorescent signal from one nucleic acid sample is compared to the fluorescent signal from the other nucleic acid sample, and the relative signal intensity determined.
[00309] Methods for analysing the data collected by fluorescence detection are known in the art. Data analysis includes the steps of determining fluorescent intensity as a function of substrate position from the data collected, removing outliers, i.e., data deviating from a predetermined statistical distribution, and calculating the relative binding affinity of the targets from the remaining data. The resulting data may be displayed as an image with the intensity in each region varying according to the binding affinity between targets and probes.
[00310] Nucleic acid analysis via microchip technology is also applicable to the present invention. In this technique, thousands of distinct oligonucleotide probes can be applied in an array on a silicon chip. A nucleic acid to be analyzed is fluorescently labeled and hybridized to the probes on the chip. It is also possible to study nucleic acid-protein interactions using these nucleic acid microchips. Using this technique one can determine the presence of mutations, sequence the nucleic acid being analyzed, or measure expression levels of a gene of interest. The method is one of parallel processing of many, even thousands, of probes at once and can tremendously increase the rate of analysis.
[00311] Alteration of mRNA transcription can be detected by any techniques known to persons of ordinary skill in the art. These include Northern blot analysis, PCR amplification and RNase protection. Diminished mRNA transcription indicates an alteration of the sequence.
[00312] The array/chip technology has already been applied with success in numerous cases.
[00313] For example, the screening of mutations has been undertaken in the BRCA 1 gene, in S. cerevisiae mutant strains, and in the protease gene of HIV-1 virus (Hacia et al., 1996; Shoemaker et al., 1996; Kozal et al., 1996). Chips of various formats for use in detecting SNPs can be produced on a customized basis.
[00314] An array-based tiling strategy useful for detecting SNPs is described in EP 785280. Briefly, arrays may generally be "tiled" for a large number of specific polymorphisms. "Tiling" refers to the synthesis of a defined set of oligonucleotide probes that are made up of a sequence complementary to the target sequence of interest, as well as preselected variations of that sequence, e.g., substitution of one or more given positions with one or more members of the basis set of monomers, i.e., nucleotides. Tiling strategies are further described in PCT application No. WO 95/11995. In some embodiments, arrays are tiled for a number of specific SNPs. In particular, the array is tiled to include a number of detection blocks, each detection block being specific for a specific SNP or a set of SNPs. For example, a detection block may be tiled to include a number of probes that span the sequence segment that includes a specific SNP. To ensure probes that are complementary to each allele, the probes are synthesized in pairs differing at the SNP position. In addition to the probes differing at the SNP position, monosubstituted probes are also generally tiled within the detection block. Such methods can readily be applied to the SNP information disclosed herein.
[00315] These monosubstituted probes have bases at and up to a certain number of bases in either direction from the polymorphism, substituted with the remaining nucleotides (selected from A, T, G, C and U). Typically, the probes in a tiled detection block will include substitutions of the sequence positions up to and including those that are 5 bases away from the SNP. The monosubstituted probes provide internal controls for the tiled array, to distinguish actual hybridization from artificial cross-hybridization. Upon completion of hybridization with the target sequence and washing of the array, the array is scanned to determine the position on the array to which the target sequence hybridizes. The hybridization data from the scanned array is then analyzed to identify which allele or alleles of the SNP are present in the sample. Hybridization and scanning may be carried out as described in PCT application No. WO 92/10092 and WO 95/11995 and US Patent No. 5,424,186.
[00316] Thus, in some embodiments, the chips may comprise an array of nucleic acid sequences of fragments of about 15 nucleotides in length and the sequences complementary thereto, or a fragment thereof, the fragment comprising at least about 8 consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides and containing a polymorphic base. In some embodiments the polymorphic base is within 5, 4, 3, 2, or 1 nucleotides from the centre of the polynucleotide, more preferably at the centre of the polynucleotide. In other embodiments, the chip may comprise an array containing any number of polynucleotides of the present invention.
[00317] An oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/251116. In another aspect, a "gridded"array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number which lends itself to the efficient use of commercially available instrumentation.
[00318] Using such arrays, the present invention provides methods of identifying the SNPs of the present invention in a sample. Such methods comprise incubating a test sample with an array comprising one or more oligonucleotide probes corresponding to at least one SNP position of the present invention, and assaying for binding of a nucleic acid from the test sample with one or more of the oligonucleotide probes. Such assays will typically involve arrays comprising oligonucleotide probes corresponding to many SNP positions and/or allelic variants of those SNP positions, at least one of which is a SNP of the present invention.
[00319] Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or array assay formats can readily be adapted to employ the novel SNPs disclosed herein. Examples of such assays can be found in Chard, T, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (I 982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).
[00320] Multicomponent integrated systems may also be used to analyze SNPs. Such systems miniaturize and compartmentalize processes such as PCR and capillary electrophoresis reactions in a single functional device. An example of such technique is disclosed in US Patent No. 5,589,136, which describes the integration of PCR amplification and capillary electrophoresis in chips.
[00321] Integrated systems can be envisaged mainly when micro-fluidic systems are used. These systems comprise a pattern of micro-channels designed onto a glass, silicon, quartz, or plastic wafer included on a microchip. The movements of the samples are controlled by electric, electro-osmotic or hydrostatic forces applied across different areas of the microchip to create functional microscopic valves and pumps with no moving parts. Varying the voltage controls the liquid flow at intersections between the micro-machined channels and changes the liquid flow rate for pumping across different sections of the microchip.
[00322] For genotyping SNPs, the microfluidic system may integrate, for example, nucleic acid amplification, mini-sequencing primer extension, capillary electrophoresis, and a detection method such as laser induced fluorescence detection.
[00323] In a first step, the DNA samples are amplified, preferably by PCR. Then, the amplification products are subjected to automated mini-sequencing reactions using ddNTPs (specific fluorescence for each ddNTP) and the appropriate oligonucleotide mini-sequencing primers which hybridize just upstream of the targeted polymorphic base. Once the extension at the 3'end is completed, the primers are separated from the unincorporated fluorescent ddNTPs by capillary electrophoresis. The separation medium used in capillary electrophoresis can be, for example, polyacrylamide, polyethylene glycol or dextran. The incorporated ddNTPs in the single nucleotide primer extension products are identified by laser-induced fluorescence detection. This microchip can be used to process at least 96 to 384 samples, or more, in parallel.
3.5 Extension based techniquesfor the detection ofpolymorphisms
[00324] Extension based techniques for detecting polymorphisms within a nucleotide sequence can include, but are not restricted to allele-specific amplification, also known as the amplification refractory mutation system (ARMS) as disclosed in European Patent Application Publication No. 0332435 and in Newton et al., 1989, and cloning of polymorphisms (COPS) as contemplated by Gibbs et al. 1989.
[00325] The extension-based technique, ARMS, uses allele specific oligonucleotide (ASO) PCR primers for genotyping. In this approach, one of the two oligonucleotide primers used for
PCR is designed to bind to the polymorphic site, most commonly with the 3' end of the primer targeting the site. Under carefully controlled conditions (annealing temperature, magnesium concentration etc.), amplification only takes place if the nucleotide at the 3' end of the PCR primer is complementary to the base at the polymorphic site, with a mismatch being "refractory" to amplification.
[00326] A variation of the ARMS approach, termed mutagenically separated PCR (MS PCR), comprises two ARMS primers of different lengths, each specific for different polymorphisms at a site. This method yields PCR products of different lengths for the different polymorphisms.
3.6 Ligation based assaysfor detecting polymorphisms
[00327] Another typical method of SNP detection encompasses the oligonucleotide ligation assay. A number of approaches make use of DNA ligase, an enzyme that can join two adjacent oligonucleotides hybridized to a DNA template. The specificity of the approach comes from the requirement for a perfect match between the hybridized oligonucleotides and the DNA template at the ligation site. In the oligonucleotide ligation assay (OLA), or ligase chain reaction (LCR) assay the sequence surrounding the mutation site is first amplified, and one strand serves as a template for three ligation probes, two of these are allele specific oligonucleotides (ASO) and the third a common probe. Numerous approaches can be used for the detection of the ligated products. For example, the two ASOs can be differentially labeled with fluorescent or hapten labels and ligated products detected by fluorimetric or colorimetric enzyme-linked immunosorbent assays, respectively. For electrophoresis-based systems, use of mobility modifier tags or variation in probe lengths coupled with fluorescence detection enables the multiplex genotyping of several single nucleotide substitutions in a single tube. When used on arrays, ASOs can be spotted at specific locations or addresses on a chip. PCR amplified DNA can then be added and ligation to labeled oligonucleotides at specific addresses on the array can be measured.
3.7 Signal generatingpolymorphism detection assays
[00328] In some embodiments, fluorescence resonance energy transfer (FRET) is contemplated as a method to identify a polymorphism within the 16S rRNA gene. FRET occurs due to the interaction between the electronic excited states of two dye molecules. The excitation is transferred from one (the donor) dye molecule to the other (the acceptor) dye molecule without emission of a photon. This is distance-dependent, that is the donor and the acceptor dye must be in close proximity. The hybridization probe system consists of two oligonucleotides labeled with fluorescent dyes. The hybridization probe pair is designed to hybridize to adjacent regions on the target DNA. Each probe is labeled with a different marker dye. Interaction of the two dyes can only occur when both are bound to their target. The donor probe is labeled with fluorophore at the 3' end and the acceptor probe at the 5' end. During PCR, the two different oligonucleotides hybridize to adjacent regions of the target DNA such that the fluorophores, which are coupled to the oligonucleotides, are in close proximity in the hybrid structure. The donor fluorophore (F) is excited by an external light source, and then passes part of its excitation energy to the adjacent acceptor fluorophore (F2). The excited acceptor fluorophore (F2) emits light at a different wavelength which can then be detected and measured for molecular proximity.
[00329] In other embodiments, the MagSNiPer method, based on single base extension, magnetic separation, and chemiluminescence provides a further method for SNP identification in a nucleotide sequence. Single base nucleotide extension reaction is performed with a biotinylated primer whose 3' terminus is contiguous to the SNP site with a tag-labeled ddNTP. Then the primers are captured by magnetic-coated beads with streptavidin, and unincorporated labeled ddNTP is removed by magnetic separation. The magnetic beads are incubated with anti-tag antibody conjugated with alkaline phosphatase. After the removal of excess conjugates by magnetic separation, SNP typing is performed by measuring chemilummescence. The incorporation of labeled ddNTP is monitored by chemilummescence induced by alkaline phosphatase.
[00330] In some embodiments, fluorescence polarization provides a method for identifying polymorphisms within a nucleotide sequence. For example, amplified DNA containing a polymorphic is incubated with oligonucleotide primers (designed to hybridize to the DNA template adjacent to the polymorphic site) in the presence of allele-specific dye-labeled dideoxyribonucleoside triphosphates and a commercially available modified Taq DNA polymerase. The primer is extended by the dye -terminator specific for the allele present on the template, increasing approximately 10-fold the molecular weight of the fluorophore. At the end of the reaction, the fluorescence polarization of the two dye-terminators in the reaction mixture is analyzed directly without separation or purification. This homogeneous DNA diagnostic method is shown to be highly sensitive and specific and is suitable for automated genotyping of large number of samples.
[00331] In other embodiments, surface enhanced Raman scattering can be used as a method for detecting and identifying single base differences in double stranded DNA fragments (see Chumanov, G 1999). SERS has also been used for single molecule detection (Kneipp, K, 1997). SERS results in strongly increased Raman signals from molecules that have been attached to nanometer sized metallic structures.
[00332] Illustrative examples include a genotyping method discussed by Xiao and Kwok 2003 based on a primer extension assay with fluorescence quenching as the detection. The template-directed dye-terminator incorporation with fluorescence quenching detection (FQ TDI) assay is based on the observation that the intensity of fluorescent dye R110- and R6G labeled acycloterminators is universally quenched once they are incorporated onto a DNA oligonucleotide primer. By comparing the rate of fluorescence quenching of the two allelic dyes in real time, the frequency of SNPs in DNA samples can be measured. The kinetic FQ TDI assay is highly accurate and reproducible both in genotyping and in allele frequency estimation.
3.8 High-resolutionmelt analysis
[00333] In particular embodiments, the methods of the present invention utilise high resolution melting (HRM) analysis for classifying and/or identifying bacterial or bacterium in a sample based on the SNP(s) described herein within the 16S rRNA gene, within the 16S rRNA molecule or within a DNA copy thereof.
[00334] HRM is based upon the accurate monitoring of changes in fluorescence as a PCR product (i.e., amplicon) stained with an intercalating fluorescent dye is heated through its melting temperature (Tm). In contrast to traditional melting, the information in HRM analysis is contained in the shape of the melting curve, rather than just the calculated T., so HRM may be considered a form of spectroscopy. HRM analysis is a single step and closed tube method, the amplification and melting can be run as a single protocol on a real-time PCR machine.
[00335] In embodiments of the present invention, the methods utilise an amplification primer pair that selectively hybridize to a target polynucleotide containing one or more of the SNPs as described herein. The amplification reaction mixture contains the fluorescent dye, which is incorporated into the resulting amplicon.
[00336] The resulting amplicon is then subjected to HRM with incremental increases in temperature (i.e., 0.01-0.5°C) ranging from about 50°C to about 95°C. At some point during this process, the melting temperature of the amplicon is reached and the two strands of DNA separate or "melt" apart.
[00337] The HRM is monitored in real-time using the fluorescent dye incorporated into the amplicon. The level of fluorescence of the dye is monitored as the temperature increases with the fluorescence reducing as the amount of double stranded DNA reduces. Changes in fluorescence and temperature can be plotted in a graph known as a melt curve.
[00338] As a skilled addressee will understand, the Tm of the amplicon at which the two DNA strands separate is predictable, being dependent on the sequence of the nucleotide bases forming the amplicon. Accordingly, it is possible to differentiate between amplicons including an amplicon containing a polymorphism (i.e., a SNP or SNPs) as the melt curves will appear different. Indeed, in some embodiments, it is possible to differentiate between amplicons containing the same polymorphism based on differences in the surrounding DNA sequences.
[00339] HRM curves can be discriminated from one another by many different strategies. For example, in many cases, HRM curves can be discriminated on the basis of obvious differences in curve shape and/or on the basis of T with a difference of 0.2°C being regarded as significant. In other cases, a difference graph analysis can be used in which a defined curve is used as a baseline with other normalised curves being plotted in relation to the baseline (see Price, E.P. et al. 2007). In yet other cases, a difference graph-based method can be used involving deriving the 3rd and 97th centiles from the mean ±1.96 standard deviations for the fluorescence at every temperature (see Andersson, P. et al., 2009; and Merchant-Patel, S. et al. 2008).
4. Tools, Reagents, Primers, Probes, Kits and Processing Systems
[00340] The specification explains how various SNPs can be used as 'tools' for identifying, partially identifying or classifying a bacterium, yeast organism or filamentous fungi, or diagnosing a bacterial, yeast organism or filamentous fungi infection. This SNP finding enables the inventors to develop gene/allele-based and gene product-based probes, tools, reagents, methods and assays for identifying, partially identifying or classifying a bacterium, yeast organism or filamentous fungi, or diagnosing a bacterial, yeast organism or filamentous fungi infection.
[00341] One of skill in the art could readily design, produce or manufacture a wide range of gene/allele-based and gene product-based probes, tools, reagents, methods and assays based on the information provided in the specification and especially in Tables 1 to 9 and 11.
[00342] Generally speaking, such probes, tools or reagents based on or developed in view of the SNPs outlined in the present specification may, for example, specifically bind, detect, identify, characterise or quantify the gene or part of the gene, the RNA gene product or part of the RNA gene product, or other gene products or parts thereof.
[00343] Generally speaking, such probe, tool or reagent can be for detection of a polymorphism for example at the genomic level, or at the transcription level.
[00344] Generally speaking, such probe, tool or reagent can also be an antibody or other type of molecule or chemical entity capable of detecting the gene or gene product (such as RNA).
[00345] More specifically, probes, tools and reagents may include, but are not limited to, the following: 1. An isolated, purified, synthetic or recombinant form of 16S rRNA, 16S rDNA, 18S rRNA or 18S rDNA, or a fragment thereof, including a fragment containing a SNP of interest - single stranded or double stranded. 2. A non-naturally occurring polynucleotide, recombinant polynucleotide, oligonucleotide or cDNA form of 16S rRNA, 16S rDNA, 18S rRNA or 18S rDNA, or a fragment thereof, including a fragment containing a SNP of interest - single stranded or double stranded. 3. An expression vector, recombinant cell or biological sample comprising the nucleic acid or polynucleotide of 1 or 2.
[00346] The probe, tool or reagent can be, but is not limited to, an antibody or other type of molecule or chemical entity capable of detecting the gene or gene product (RNA or polypeptide).
[00347] The at least one probe, tool or reagent can be any number or combination of the above, and the number and combination will depend on the desired result to be achieved - eg. detection of a polymorphism at the genomic level (genotyping), at the RNA level.
[00348] All the essential materials and reagents required for detecting one or more SNPs in the 16S rRNA gene or 18S rRNA gene according to the invention may be assembled together in a kit. The kits may also optionally include appropriate reagents for detection of labels, positive and negative controls, fluorescent dyes, washing solutions, blotting membranes, microtitre plates, dilution buffers and the like. For example, a nucleic acid-based detection kit for the identification of polymorphisms may include one or more of the following: (i) nucleic acid from a Gram-positive cell and/or Gram-negative cell (which may be used as a positive control); and (ii) a primer and/or probe that specifically hybridizes to at least a portion of the 16S rRNA gene or 18S rRNA gene containing the SNP position(s) to be analysed, and optionally one or more other markers, at or around the suspected SNP site. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (Reverse Transcriptase, Taq, SequenaseTM DNA ligase etc. depending on the nucleic acid amplification technique employed), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification. Such kits also generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe. The kit can also feature various devices and reagents for performing one of the assays described herein; and/or printed instructions for using the kit to identify the presence of a SNP as defined herein.
[00349] In some embodiments, the methods described generally herein are performed, at least in part, by a processing system, such as a suitably programmed computer system. A stand-alone computer, with the microprocessor executing applications software allowing the above-described methods to be performed, may be used. Alternatively, the methods can be performed, at least in part, by one or more processing systems operating as part of a distributed architecture. For example, a processing system can be used to detect the presence of an SNP at a position by detecting the hybridization of a probe to a nucleic acid molecule. A processing system also can be used to determine the Gram status or identity or grouping of a bacterium on the basis of detection of one or more SNPs. In some examples, commands inputted to the processing system by a user may assist the processing system in making these determinations.
[00350] In one example, a processing system includes at least one microprocessor, a memory, an input/output device, such as a keyboard and/or display, and an external interface, interconnected via a bus. The external interface can be utilised for connecting the processing system to peripheral devices, such as a communications network, database, or storage devices. The microprocessor can execute instructions in the form of applications software stored in the memory to allow the SNP detection and/or microorganism identification or classification process to be performed, as well as to perform any other required processes, such as communicating with the computer systems. The application software may include one or more software modules, and may be executed in a suitable execution environment, such as an operating system environment, or the like.
4.1 Primers, Probes, Kits and Processing Systems for the 16S rRNA gene or the 18S rRNA gene
[00351] The present invention provides probes and primers that may be used in the methods described herein to determine SNPs at one or more positions of the 16S rRNA gene or 18S rRNA gene so as to classify and/or identify bacteria or bacterium, yeast organism or filamentous fungi in a sample.
[00352] The primers and probes of the present invention hybridize to at least a portion of the 16S rRNA gene or the 18S rRNA gene (or 16S rRNA molecules or DNA copies thereof or 18S rRNA molecules or DNA copies thereof) containing the SNP position(s). For example, the primers may hybridize to a sequence flanking one or more SNPs, and the probe may hybridize to a sequence that includes one or more SNPs. It is well within the skill of a skilled artisan to design appropriate primers and probes for use in the methods of the present invention, based on the known sequences of the 16S rRNA gene or the 18S rRNA gene.
[00353] Non-limiting examples of primers and probes that are useful for the methods of the present invention, in which SNPs in the 16S rRNA of bacterial species at positions corresponding to positions 273, 378, 412, 440, 488, 647 and/or 653 of the 16S rRNA gene set forth in SEQ ID NO:1 are analysed, include those described in Example 1.
[00354] For example, to detect SNPs at position 273 an exemplary forward primer includes CCTCTTGCCATCGGATGTG (SEQ ID NO:16) and exemplary reverse primers include CCAGTGTGGCTGGTCATCCT (SEQ ID NO:17), CGATCCGAAAACCTTCTTCACT (SEQ ID NO:20), CTATGCATCGTTGCCTTGGTAA (SEQ ID NO:22), TGATGTACTATTAACACATCAACCTTCCT (SEQ ID NO:26), AACGCTCGGATCTTCCGTATTA (SEQ ID NO:27), CGCTCGCCACCTACGTATTAC (SEQ ID NO:28), CGTAGTTAGCCGTCCCTTTCTG (SEQ ID NO:30), GGAATTCTACCCCCCTCTACGA (SEQ ID NO:34), and GGAATTCTACCCCCCTCTACAAG (SEQ ID NO:35).
[00355] To detect SNPs at position 378, exemplary forward primers include CCTCTTGCCATCGGATGTG (SEQ ID NO:16), CCTACGGGAGGCAGCAGTAG (SEQ ID NO:18), and GGGAGGCAGCAGTAGGGAAT (SEQ ID NO:19); and exemplary reverse primers include CGATCCGAAAACCTTCTTCACT (SEQ ID NO:20), CTATGCATCGTTGCCTTGGTAA (SEQ ID NO:22), TGATGTACTATTAACACATCAACCTTCCT (SEQ ID NO:26), AACGCTCGGATCTTCCGTATTA (SEQ ID NO:27), CGCTCGCCACCTACGTATTAC (SEQ ID NO:28), CGTAGTTAGCCGTCCCTTTCTG (SEQ ID NO:30), GGAATTCTACCCCCCTCTACGA (SEQ ID NO:34), and GGAATTCTACCCCCCTCTACAAG (SEQ ID NO:35).
[00356] To detect SNPs at position 412, exemplary forward primers include CCTCTTGCCATCGGATGTG (SEQ ID NO:16), CCTACGGGAGGCAGCAGTAG (SEQ ID NO:18), GGGAGGCAGCAGTAGGGAAT (SEQ ID NO:19), and AAGACGGTCTTGCTGTCACTTATAGA (SEQ ID NO:21); and exemplary reverse primers include CTATGCATCGTTGCCTTGGTAA (SEQ ID NO:22), TGATGTACTATTAACACATCAACCTTCCT (SEQ ID NO:26), AACGCTCGGATCTTCCGTATTA (SEQ ID NO:27), CGCTCGCCACCTACGTATTAC (SEQ ID NO:28), CGTAGTTAGCCGTCCCTTTCTG (SEQ ID NO:30), GGAATTCTACCCCCCTCTACGA (SEQ ID NO:34), and GGAATTCTACCCCCCTCTACAAG (SEQ ID NO:35).
[00357] To detect SNPs at position 440, exemplary forward primers include CCTCTTGCCATCGGATGTG (SEQ ID NO:16), CCTACGGGAGGCAGCAGTAG (SEQ ID NO:18), GGGAGGCAGCAGTAGGGAAT (SEQ ID NO:19), AAGACGGTCTTGCTGTCACTTATAGA (SEQ ID NO:21), TGCCGCGTGAATGAAGAA (SEQ ID NO:23), GCGTGAAGGATGAAGGCTCTA (SEQ ID NO:24), and TGATGAAGGTTTTCGGATCGT (SEQ ID NO:25); and exemplary reverse primers include TGATGTACTATTAACACATCAACCTTCCT (SEQ ID NO:26), AACGCTCGGATCTTCCGTATTA (SEQ ID NO:27), CGCTCGCCACCTACGTATTAC (SEQ ID NO:28), CGTAGTTAGCCGTCCCTTTCTG (SEQ ID NO:30), GGAATTCTACCCCCCTCTACGA (SEQ ID NO:34), and GGAATTCTACCCCCCTCTACAAG (SEQ ID NO:35).
[00358] To detect SNPs at position 488, exemplary forward primers include CCTCTTGCCATCGGATGTG (SEQ ID NO:16), CCTACGGGAGGCAGCAGTAG (SEQ ID NO:18), GGGAGGCAGCAGTAGGGAAT (SEQ ID NO:19), AAGACGGTCTTGCTGTCACTTATAGA (SEQ ID NO:21), TGCCGCGTGAATGAAGAA (SEQ ID NO:23), GCGTGAAGGATGAAGGCTCTA (SEQ ID NO:24), TGATGAAGGTTTTCGGATCGT (SEQ ID NO:25), and GTTGTAAGAGAAGAACGAGTGTGAGAGT (SEQ ID NO:29); and exemplary reverse primers include CGTAGTTAGCCGTCCCTTTCTG (SEQ ID NO:30), GGAATTCTACCCCCCTCTACGA (SEQ ID NO:34), and GGAATTCTACCCCCCTCTACAAG (SEQ ID NO:35).
[00359] To detect SNPs at positions 647 and/or 653, exemplary forward primers include CCTCTTGCCATCGGATGTG (SEQ ID NO:16), CCTACGGGAGGCAGCAGTAG (SEQ ID
NO:18), GGGAGGCAGCAGTAGGGAAT (SEQ ID NO:19), AAGACGGTCTTGCTGTCACTTATAGA (SEQ ID NO:21), TGCCGCGTGAATGAAGAA (SEQ ID NO:23), GCGTGAAGGATGAAGGCTCTA (SEQ ID NO:24), TGATGAAGGTTTTCGGATCGT (SEQ ID NO:25), GTTGTAAGAGAAGAACGAGTGTGAGAGT (SEQ ID NO:29), GCGGTTTGTTAAGTCAGATGTGAA (SEQ ID NO:31), GGTCTGTCAAGTCGGATGTGAA (SEQ ID NO:32), and TCAACCTGGGAACTCATTCGA (SEQ ID NO:33); and exemplary reverse primers include GGAATTCTACCCCCCTCTACGA (SEQ ID NO:34), and GGAATTCTACCCCCCTCTACAAG (SEQ ID NO:35).
[00360] Similarly, non-limiting examples of primers and probes that are useful for the methods of the present invention, in which SNPs in the 16S rRNA gene or 16S rRNA of bacterial species at positions corresponding to positions corresponding to positions 746, 764, 771, or 785 of the 16S rRNA gene as set forth in SEQ ID NO:43 (or positions 737, 755, 762, or 776 of the 16S rRNA gene as set forth in SEQ ID NO:1) are analysed, include those described in Table 8.
[00361] Similarly, non-limiting examples of primers and probes that are useful for the methods of the present invention, in which SNPs in the 18S rRNA gene or 18S rRNA of bacterial species at positions 343, 371, 388, 416, and 467 of the 18S rRNA gene set forth in SEQ ID NO: 37 are analysed, include those described in Table 9.
5. Applications of the methods of the present invention
[00362] The methods of the present invention are useful for classifying and/or identifying bacteria, yeast organism or filamentous fungi in a sample, such as a sample from a subject or an environmental sample such as a soil or water sample or a sample taken from the surface of equipment or instruments (e.g. medical or surgical instruments) or a work surface. Such classification or identification can then be used to determine a course of treatment to remove, eradicate or reduce the number of bacteria, yeast organism or filamentous fungi. Any two or more of the methods of the present invention can be combined. For example, nucleic acid from a sample can be analysed for the presence of SNPs in a 16S rRNA gene using the methods of the present invention. This can be done so as to determine whether Gram-positive bacteria or Gram-negative bacteria are present in the sample. The bacteria can be further grouped or the identity of the bacterium may also be determined or narrowed down to one of a few possibilities. For example, as would be apparent from the disclosure above, SNPs at positions corresponding to positions 273, 378, 412, 440, 488, 647 and 653 of the 16S rRNA gene set forth in SEQ ID NO:1 can be assessed so as to classify or even identify a bacterium in a sample.
[00363] Subjects with infections or suspected infections often present to clinicians in clinics, emergency rooms, general wards and intensive care units. Such patients often have non diagnostic clinical signs of abnormal temperature, increased heart and respiratory rates and abnormal white cells counts. A clinician must decide whether the patient has an infection or not, the severity of the infection, whether to admit the patient to hospital (if not already in hospital), the source of infection, whether to use antibiotics, and if so, the type, route and dose of antibiotics. The presence of an infection in a patient has most typically been assessed by taking a sample from the patient and growing an organism in culture broth. Once an organism has grown it can be Gram stained and identified. However, in many infected patients (>50) it is not possible to culture an organism. Without an identified organism, a clinician must rely on clinical judgment and the use of broad spectrum antibiotics often in combinations. The indiscriminate use of broad-spectrum antibiotics, without knowledge of the pathogenic organism's identity or sensitivity, results in the development of antibiotic resistance, overuse of antibiotics, and potentially toxic side effects in patients. Blood culture is a sensitive method (1 100 cfu/mL) but only when the blood sample taken contains a viable organism, which is not always the case.
[00364] Thus, the methods of the present invention are particularly useful in assisting clinicians in determining whether the subject has an infection and if so, an appropriate course of treatment based on the classification of the bacteria, yeast organism or filamentous fungi causing the infection.
[00365] Furthermore, the methods of the present invention facilitate discrimination of Gram-positive and Gram-negative organisms within hours of taking a whole blood from a subject. The methods of the present invention also can be performed in a time-efficient manner, so that the results are available to the clinician within hours rather than days. Such attributes allow a clinician to sensitively detect the presence of a bacterium, yeast organism or filamentous fungi and to make an informed decision on treatment (such as the use of antibiotics specific to the Gram status or further grouping or identification of the bacterium). These improvements can result in a reduced number of patients admitted to hospital unnecessarily, sensitive detection of bacteria, yeast organisms and filamentous fungi, severity of infection assessed on load (and other factors), reduced use of broad-spectrum antibiotics/medicines, reduced patient time on broad spectrum antibiotics, reduced toxicity from antibiotics/medicines, reduced development of resistance to medicines (especially antibiotic resistance).
[00366] The present invention also extends to diagnosing a bacterial, yeast organism or filamentous fungi infection in a subject, and the management of the infection following a positive diagnosis. The methods described herein that analyse one or more SNPs within a 16S rRNA or 18S rRNA can be used to determine whether a subject has a bacterial, yeast organism or filamentous fungi infection and/or identify the group or species of bacteria, yeast organism or filamentous fungi in the sample. The methods described herein can be further used to classify a bacteria as Gram-positive or Gram-negative.
5.1 Management and therapy
[00367] Based on the results of the methods of the present invention, the subject can be appropriately managed and administered therapy where required. For example, the management of a bacterial infection can include, for example, administration of therapeutic agents such as a therapeutically effective course of antibiotics.
[00368] Typically, therapeutic agents will be administered in pharmaceutical (or veterinary if the subject is a non-human subject) compositions together with a pharmaceutically acceptable carrier and in an effective amount to achieve their intended purpose. The dose of active compounds administered to a subject should be sufficient to achieve a beneficial response in the subject over time such as a reduction in, or relief from, the symptoms of the infection, and/or the reduction or elimination of the bacteria from the subject. The quantity of the pharmaceutically active compounds(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the active compound(s) for administration will depend on the judgment of the practitioner. In determining the effective amount of the active compound(s) to be administered in the treatment or prevention of the bacterial infection, the practitioner may evaluate severity of infection, and severity of any symptom associated with the infection including, inflammation, blood pressure anomaly, tachycardia, tachypnoea, fever, chills, vomiting, diarrhoea, skin rash, headaches, confusion, muscle aches and seizures. In any event, those of skill in the art may readily determine suitable dosages of the therapeutic agents and suitable treatment regimens without undue experimentation.
[00369] The therapeutic agents may be administered in concert with adjunctive (palliative) therapies to increase oxygen supply to major organs, increase blood flow to major organs and/or to reduce the inflammatory response. Illustrative examples of such adjunctive therapies include non steroidal-anti inflammatory drugs (NSAIDs), intravenous saline and oxygen.
[00370] Reference throughout this specification to 'one embodiment' or 'an embodiment' means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the phrases 'in one embodiment' or 'in an embodiment' in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more combinations.
[00371] In compliance with the statute, the invention has been described in language more or less specific to structural or methodical features. It is to be understood that the invention is not limited to specific features shown or described since the means herein described comprises preferred forms of putting the invention into effect. The invention is, therefore, claimed in any of its forms or modifications within the proper scope of the appended claims (if any) appropriately interpreted by those skilled in the art.
[00372] The disclosure of every patent, patent application and publication cited herein is hereby incorporated herein by reference in its entirety.
[00373] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.
EXAMPLES
EXAMPLE 1
[00374] The aim of this experiment was to differentiate 15 of the most prevalent bacterial species frequently isolated from patients diagnosed with sepsis using the 16S rRNA SNP-HRM assay of the present invention.
Experimental Procedures
[00375] In total, the following 15 bacterial species were tested: Acinetobacter calcoaceticus; Enterobacteraerogenes; Enterobacter cloacae; Enterococcusfaecalis; Enterococcusfaecium; Escherichiacoli; Klebsiellapneumoniae; Proteusmirabilis;Pseudomonas aeruginosa;Serratia marcescens; Staphylococcus aureus; Staphylococcus epidermidis; Streptococcus agalactiae; Streptococcus pneumoniae; and Streptococcus pyogenes.
[00376] The bacterial species were cultured in Brain Heart Infusion broth overnight at 37C whereafter genomic DNA was extracted from each isolate using QIAgen DNeasy Blood and Tissue Kit (Qiagen, Australia).
[00377] 16S rDNA-SNP primers (of SEQ ID NOs:16-35, made by Sigma Aldrich, Australia) were designed to amplify the regions encompassing the seven SNPs designated as follows: SNP273, SNP378, SNP412, SNP440, SNP488, SNP647 and SNP653 of the 16S rRNA gene as set forth in SEQ ID NO:1. PCR product sizes ranged from 79bp to 96bp.
[00378] Real-Time PCR followed by HRM analysis was used to differentiate the 15 bacterial species. The Real-Time PCR HRM process was: 1 pl of extracted DNA (1-3 ng) was added to 19 pl of reaction mastermix containing 10pl of the 2X SYBR green PCR Mastermix (Invitrogen, Australia) and 8 pmol of each primer. Temperature cycling for these reactions were as follows: 50°C for 2min, 95°C for 2min, followed by 40 cycles of 95°C for 15s, 52°C for 20s, and 72°C for 35s, hold at 72°C for 2min, hold at 50°C for 20s, HRM: ramp from 65°C to 95°C rising by 0.05°C (Rotor-Gene 6000, Qiagen, Australia).
[00379] The Rotor-Gene 6000 software (version 1.7.34 or 1.7.87) was used to analyse the HRM data in multiple ways. A normalised raw melt curve depicts decreasing fluorescence versus increasing temperature, and the difference curve, which displays a user-defined curve as the baseline (i.e., the x-axis), and depicts other normalised curves in relation to that baseline. Criteria for calling melting curves as "same" or "different" using difference graphs have been developed and published previously by the inventor and her co-workers (see, e.g., Stephens, A.J., et al. 2008; Merchant-Patel, S., et al. 2010).
[00380] There are two ways that HRM curve plots can discriminate between samples. The shape of the melt curve indicates the details in the shape of the curve, and the curve shift indicates a thermal (temperature) offset of a curve from other curves.
[00381] The software allows HRM melt curve analysis using either as normalised melt curves or as difference curves as generally described above. Normalisation curve analysis allows all the HRM curves to be compared with the same starting and ending fluorescent signal level to aid interpretation and analysis. The difference curve analysis displays differences between the melt curves of each sample and a given control.
[00382] In this experiment, both the shape and shift approach was used to discriminate the HRM curves for each bacterial species tested. For determining the shift in the melting temperature for each respective bacterial species, a melting temperature difference of 0.2°C was regarded as a significant difference between each bacterial species' melt curves.
[00383] For determining the shape differences between bacterial species, the difference curve analysis was used. An amplitude difference of >5 normalised fluorescence units is indicative that different bacterial species melt curves are different to the comparator species. Hence these differences in the shape of the curve indicate differences in the DNA sequences of each respective bacterial species.
Results
[00384] Figures 2 to 11 depict normalised and difference melting curves plots used to differentiate the 15 bacterial species tested in this example.
[00385] Each of the 15 bacterial species was designated with species specific genotypes in the HRM analysis setting of the Rotor-Gene 6000 software (version 1.7.34 or 1.7.87). For comparison of the bacterial species difference curves, Escherichia coli was used as the "calibrator" or "reference" (indicated as "0" on the Y-axis of the plot shown in Figure 3. As is shown in Figure 3, the 14 other bacterial species all showed curves that differ away from the "0" line of Escherichiacoli. Another example of species differentiation is shown in Figure 5, where Staphylococcus aureus is selected as the "calibrator" or "reference" (at the "0" position on the Y-axis) and as shown Staphylococcus epidermidis has a complete separate curve.
[00386] The melt curve specificity for the bacteria in urine and plasma is provided in Table 11.
Table 11 - Bacterial speciation in urine and plasma: Melt curve specificity Name Tm' SNP* no. E. coli 77.075 1 E. cloacae 79.050 1 S. marcescens 77.810 1 E. aerogenes 78.250 1 K. pneumoniae 78.435 1
E. faecalis 83.850 2 E. faecium 83.475 2 S. agalactiae 83.925 2 S.pyogenes 84.825 2
S.aureus 81.485 3 S. epidermidis 81.700 3
S. pneumoniae 78.225 4 $Tm: PCR product melting temperature; *SNP: Single nucleotide polymorphism. p-value SNP1vsSNP2: <0.0001; p-value SNP2vsSNP3: 0.002; p-value SNP3vsSNP4: 0.0005.
Discussion
[00387] This example demonstrates the utility of applying only seven highly discriminating SNPs to differentiate between 15 different bacterial species known to cause life-threatening diseases such as sepsis. The results also indicate that the method of the present invention is highly specific and rapid, both of which are important requirements for a DNA diagnostic assay. This method accurately determines whether two isolates are the same or different based on the DNA melt curves of the PCR products encompassing the highly discriminatory SNPs. The interrogation of these genetic targets means that this approach is especially amenable to adaption to emerging technologies such as "lab-on-chip" devices and dedicated, fully automated real-time PCR machines. Combined with rapidly advancing innovations in microfluidics, the methods of the present invention are suitable for transfer onto devices suitable for "point-of-care" diagnostics.
EXAMPLE 2
[00388] To demonstrate the utility of the present invention, two hundred blood culture positive patient samples were assessed by both standard clinical microbiology and by the methods of the present invention.
[00389] The standard clinical microbiology tests were performed by a routine blood culture procedure in the laboratory utilising the BacTAlert system followed by the MALDI biotyper method for bacterial species identification. This involved entering the BacTAlert blood culture bottles into an automated, continuous-monitoring incubation that are incubated for 5-7 days. Once the blood culture bottle is flagged as positive (a minimum of 12 hours incubation), the bottle is removed from the BacTAlert instrument and an aliquot of the growth medium is removed and sub-cultured onto bacterial culture agar plates. The agar plates are incubated at 37 °C for at least 4 hours, or until visible growth appears. Thereafter, a single bacterial colony is placed onto the target plate and a matrix solution is added. The plate is inserted into the biotyper instrument and a MALDI-TOF spectrum is generated by the software. The spectrum is matched against a reference library to provide bacterial identification. The total time for this process is around 16-18 hours. The standard clinical microbiology tests were performed by Pathology Queensland.
[00390] The method used according to the present invention was to collect blood culture liquid (lmL) from 100 blood culture-negative samples after 5 days of incubation on a BacTAlert blood culture machine and stored at 4°C until extracted. Blood culture liquid (lmL) was also collected from 200 blood-culture positive samples by staff at the Diagnostic Microbiology Department, Pathology Queensland and stored at 4°C until DNA was extracted. Microbial DNA was isolated from all samples (blood-culture negative and blood-culture positive) using the MolYsisTM Complete 5 kit (Molzym Life Science, Germany) which enables host DNA removal, pathogen enrichment and DNA extraction from lmL of sample.
[00391] All 300 DNA extractions were subjected to testing using a real-time PCR fonnat as follows: One microliter of extracted DNA (1 to 3 ng) was added to 19pl of reaction mastermix containing 10 pl of the 2x SYBR green PCR Mastermix (Life Technologies, Australia) and 8 pmol of each primer. Temperature cycling for these reactions were as follows: 50°C for 2 min, 95°C for 2 min, followed by 40 cycles of 95°C for 15s, 52°C for 20s, and 72°C for 35s, Hold at 72°C for 2 min, Hold at 50 °C for 20s, HRM: Ramp from 65°C - 95°C rising by 0.05°C (RotorGeneQ, Qiagen, Australia). All samples were run in duplicate, including the relevant controls (No Template (NTC) and a positive control consisting of bacterial reference DNA for each bacterial species tested. The time to result was recorded as ±3.5hrs.
[00392] The results were tabulated and were correlated to blood culture microbiology results obtained from Pathology Queensland at the conclusion of the study.
Results
[00393] The restuls of the trial are provided in Table 12.
Table 12 - Correlation between method of the present invention and clinical microbiology results (Pathology QLD trial). Ten bacterial species were represented in 200 blood culture positive patient samples. Bacterial species Gram status Clinical microbiology Method of result - samples positive present invention Staphylococcus aureus Positive 56 55 Staphylococcus Positive 55 54 epidermidis Enterococcusfaecalis Positive 11 11 Escherichiacoli Negative 50 50 Enterobactercloacae Negative 5 5 Klebsiellapneumoniae Negative 4 4 Serratiamarcescens Negative 6 6 Streptococcus agalactiae Positive 7 6 Streptococcus pyogenes Positive 3 3 Streptococcus pneumoniae Positive 3 3 Total 200 198(99% specificity)
[00394] Advantageously, the method of the present invention was able to obtain 99% specificity when compared to the clinical microbiology result (however, it is unclear for the two samples where the clinical microbiology and the method of the present invention obtained different results which method produced the incorrect result). The method of the present invention was able to obtain the result within about 3.5 hours and with minimal handling of the patient sample. In contrast, the clinical microbiology result required more significant handling of the patient sample and took about 16-18 hours to obtain.
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ggatgaccag ccacactgga ggatgaccag ccacactggaactgagacac actgagacacggtccagact ggtccagact cctacgggag cctacgggag gcagcagtgg gcagcagtgg 360 360
ggaatattgc acaatgggcg ggaatattgc acaatgggcgcaagcctgat caagcctgatgcagccatgc gcagccatgc cgcgtgtatg cgcgtgtatg aagaaggcct aagaaggcct 420 420
tcgggttgta aagtactttc tcgggttgta aagtactttcagcggggagg agcggggaggaagggagtaa aagggagtaa agttaatacc agttaatacc tttgctcatt tttgctcatt 480 gacgttaccc gcagaagaag gacgttaccc gcagaagaagcaccggctaa caccggctaactccgtgcca ctccgtgcca gcagccgcgg gcagccgcgg taatacggag taatacggag 540 540 ggtgcaagcg ttaatcggaa ggtgcaagcg ttaatcggaattactgggcg ttactgggcgtaaagcgcac taaagcgcac gcaggcggtt gcaggcggtt tgttaagtca tgttaagtca 600 600 gatgtgaaat ccccgggctc gatgtgaaat ccccgggctcaacctgggaa aacctgggaactgcatctga ctgcatctga tactggcaag tactggcaag cttgagtctc cttgagtctc 660 660 gtagaggggg gtagaattcc gtagaggggg gtagaattccaggtgtagcg aggtgtagcggtgaaatgcg gtgaaatgcg tagagatctg tagagatctg gaggaatacc gaggaatacc 720 720 ggtggcgaag gcggccccct ggtggcgaag gcggccccctggacgaagac ggacgaagactgacgctcag tgacgctcag gtgcgaaagc gtgcgaaagc gtggggagca gtggggagca 780 780 aacaggatta gataccctgg aacaggatta gataccctggtagtccacgc tagtccacgccgtaaacgat cgtaaacgat gtcgacttgg gtcgacttgg aggttgtgcc aggttgtgcc 840 840 cttgaggcgt ggcttccgga cttgaggcgt ggcttccggagctaacgcgt gctaacgcgttaagtcgace taagtcgacc gcctggggag gcctggggag tacggccgca tacggccgca 900 900 aggttaaaac tcaaatgaat aggttaaaac tcaaatgaattgacgggggc tgacgggggcccgcacaage ccgcacaagc ggtggagcat ggtggagcat gtggtttaat gtggtttaat 960 960 tcgatgcaac gcgaagaacc tcgatgcaac gcgaagaaccttacctggtc ttacctggtcttgacatcca ttgacatcca cagaactttc cagaactttc cagagatgga cagagatgga 1020 1020 ttggtgcctt cgggaactgtgagacaggtg ttggtgcctt cgggaactgt gagacaggtgctgcatggct ctgcatggct gtcgtcagct gtcgtcagct cgtgttgtga cgtgttgtga 1080 1080 aatgttgggt taagtcccgc aatgttgggt taagtcccgcaacgagcgca aacgagcgcaacccttatct acccttatct tttgttgcca tttgttgcca gcggtccggc gcggtccggc 1140 1140 cgggaactca aaggagactg cgggaactca aaggagactgccagtgataa ccagtgataaactggaggaa actggaggaa ggtggggatg ggtggggatg acgtcaagtc acgtcaagtc 1200 1200 atcatggccc ttacgaccag atcatggccc ttacgaccagggctacacac ggctacacacgtgctacaat gtgctacaat ggcgcataca ggcgcataca aagagaagcg aagagaagcg 1260 1260 acctcgcgag agcaagcgga acctcgcgag agcaagcggacctcataaag cctcataaagtgcgtcgtag tgcgtcgtag tccggattgg tccggattgg agtctgcaac agtctgcaac 1320 1320 tcgactccat gaagtcggaa tcgactccat gaagtcggaatcgctagtaa tcgctagtaatcgtggatca tcgtggatca gaatgccacg gaatgccacg gtgaatacgt gtgaatacgt 1380 tcccgggcct tgtacacacc gcccgtcaca tcccgggcct tgtacacacc gcccgtcacaccatgggagt ccatgggagt gggttgcaaa gggttgcaaa agaagtaggt agaagtaggt 1440 1440 agcttaacct tcgggagggc agcttaacct tcgggagggcgcttaccact gcttaccactttgtgattca ttgtgattca tgactggggt tgactggggt gaagtcgtaa gaagtcgtaa 1500 1500 caaggtaacc gtaggggaac ctgcggttgg atcacctcct ta caaggtaacc 1542 1542 gtaggggaac ctgcggttgg atcacctcct ta
<210> <210> 2 2 <211> <211> 1555 1555 <212> <212> DNA DNA <213> <213> Staphylococcusaureus Staphylococcus aureus
<400> <400> 2 2 ttttatggag agtttgatcctggctcagga ttttatggag agtttgatcc tggctcaggatgaacgctgg tgaacgctgg cggcgtgcct cggcgtgcct aatacatgca aatacatgca
agtcgagcga acggacgaga agtcgagcga acggacgagaagcttgcttc agcttgcttctctgatgtta tctgatgtta gcggcggacg gcggcggacg ggtgagtaac ggtgagtaac 120 120
acgtggataa cctacctata acgtggataa cctacctataagactgggat agactgggataacttcggga aacttcggga aaccggagct aaccggagct aataccggat aataccggat 180 180
aatattttga accgcatggt aatattttga accgcatggttcaaaagtga tcaaaagtgaaagacggtct aagacggtct tgctgtcact tgctgtcact tatagatgga tatagatgga 240 240
tccgcgctgc attagctagt tccgcgctgc attagctagttggtaaggta tggtaaggtaacggcttacc acggcttacc aaggcaacga aaggcaacga tgcatagccg tgcatagccg 300 300
acctgagagg gtgatcggcc acctgagagg gtgatcggccacactggaac acactggaactgagacacgg tgagacacgg tccagactcc tccagactcc tacgggaggc tacgggaggc 360 360
agcagtaggg aatcttccgc agcagtaggg aatcttccgcaatgggcgaa aatgggcgaaagcctgacgg agcctgacgg agcaacgccg agcaaccccg cgtgagtgat cgtgagtgat 420 420
gaaggtcttc ggatcgtaaa gaaggtcttc ggatcgtaaaactctgttat actctgttattagggaagaa tagggaagaa catatgtgta catatgtgta agtaactgtg agtaactgtg 480 480
cacatcttga cggtacctaa cacatcttga cggtacctaatcagaaagcc tcagaaagccacggctaact acggctaact acgtgccagc acgtgccage agccgcggta agccgcggta 540 540
atacgtaggt ggcaagcgtt atacgtaggt ggcaagcgttatccggaatt atccggaattattgggcgta attgggcgta aagcgcgcgt aagcgcgcgt aggcggtttt aggcggtttt 600 ttaagtctga tgtgaaagcc ttaagtctga tgtgaaagcccacggctcaa cacggctcaaccgtggaggg ccgtggaggg tcattggaaa tcattggaaa ctggaaaact ctggaaaact 660 660 tgagtgcaga agaggaaagt tgagtgcaga agaggaaagtggaattccat ggaattccatgtgtagcggt gtgtagcggt gaaatgcgca gaaatgcgca gagatatgga gagatatgga 720 720 ggaacaccag tggcgaaggc ggaacaccag tggcgaaggcgactttctgg gactttctggtctgtaactg tctgtaactg acgctgatgt acgctgatgt gcgaaagcgt gcgaaagcgt 780 780 ggggatcaaa caggattaga ggggatcaaa caggattagataccctggta taccctggtagtccacgccg gtccacgccg taaacgatga taaacgatga gtgctaagtg gtgctaagtg 840 840 ttagggggtt tccgccccttagtgctgcag ttagggggtt tccgcccctt agtgctgcagctaacgcatt ctaacgcatt aagcactccg aagcactccg cctggggagt cctggggagt 900 900 acgaccgcaa ggttgaaact acgaccgcaa ggttgaaactcaaaggaatt caaaggaattgacggggacc gacggggacc cgcacaagcg cgcacaagcg gtggagcatg gtggagcatg 960 960 tggtttaatt cgaagcaacgcgaagaacct tggtttaatt cgaagcaacg cgaagaaccttaccaaatct taccaaatct tgacatcctt tgacatcctt tgacaactct tgacaactct 1020 1020 agagatagag ctttcccctt agagatagag ctttccccttcgggggacaa cgggggacaaagtgacaggt agtgacaggt ggtgcatggt ggtgcatggt tgtcgtcagc tgtcgtcagc 1080 1080 tcgtgtcgtg agatgttgggttaagtcccg tcgtgtcgtg agatgttggg ttaagtcccgcaacgagcgc caacgagcgc aacccttaag aacccttaag cttagttgcc cttagttgcc 1140 1140 atcattaagt tgggcactct atcattaagt tgggcactctaagttgactg aagttgactgccggtgacaa ccggtgacaa accggaggaa accggaggaa ggtggggatg ggtggggatg 1200 1200 acgtcaaatc atcatgcccc acgtcaaatc atcatgccccttatgatttg ttatgatttgggctacacac ggctacacac gtgctacaat gtgctacaat ggacaataca ggacaataca 1260 1260 aagggcagcg aaaccgtgag aagggcagcg aaaccgtgaggtcaagcaaa gtcaagcaaatcccataaag tcccataaag ttgttctcag ttgttctcag ttcggattgt ttcggattgt 1320 1320 agtctgcaac tcgactacat agtctgcaac tcgactacatgaagctggaa gaagctggaatcgctagtaa tcgctagtaa tcgtagatca tcgtagatca gcatgctacg gcatgctacg 1380 1380 gtgaatacgt tcccgggtct gtgaatacgt tcccgggtcttgtacacacc tgtacacaccgcccgtcaca gcccgtcaca ccacgagagt ccacgagagt ttgtaacacc ttgtaacacc 1440 1440 cgaagccggt ggagtaacct cgaagccggt ggagtaaccttttaggagct tttaggagctagccgtcgaa agccgtcgaa ggtgggacaa ggtgggacaa atgattgggg atgattgggg 1500 tgaagtcgta acaaggtagc cgtatcggaa ggtgcggctg gatcacctcc tttct tgaagtcgta acaaggtage cgtatcggaa ggtgcggctg gatcacctcc tttct 1555 1555
<210> <210> 3 3 <211> <211> 1554 1554 <212> <212> DNA DNA <213> <213> Staphylococcus epidermidis Staphylococcus epidermidis
<400> <400> 3 3 ttttatggag agtttgatcctggctcagga ttttatggag agtttgatcc tggctcaggatgaacgctgg tgaacgctgg cggcgtgcct cggcgtgcct aatacatgca aatacatgca
agtcgagcga acagacgagg agtcgagcga acagacgaggagcttgcttc agcttgcttctctgacgtta tctgacgtta gcggcggacg gcggcggacg ggtgagtaac ggtgagtaac 120 120
acgtggataa cctacctata acgtggataa cctacctataagactgggat agactgggataacttcggga aacttcggga aaccggagct aaccggagct aataccggat aataccggat 180 180
aatatattga accgcatggt aatatattga accgcatggttcaatagtga tcaatagtgaaagacggttt aagacggttt tgctgtcact tgctgtcact tatagatgga tatagatgga 240 240
tccgcgccgc attagctagt tccgcgccgc attagctagttggtaaggta tggtaaggtaacggcttacc acggcttacc aaggcaacga aaggcaacga tgcgtagccg tgcgtagccg 300 300
acctgagagg gtgatcggcc acctgagagg gtgatcggccacactggaac acactggaactgagacacgg tgagacacgg tccagactcc tccagactcc tacgggaggc tacgggaggc 360 360
agcagtaggg aatcttccgc agcagtaggg aatcttccgcaatgggcgaa aatgggcgaaagcctgacgg agcctgacgg agcaacgccg agcaaccccg cgtgagtgat cgtgagtgat 420 420
gaaggtcttc ggatcgtaaa gaaggtcttc ggatcgtaaaactctgttat actctgttattagggaagaa tagggaagaa caaatgtgta caaatgtgta agtaactatg agtaactatg 480 480
cacgtcttga cggtacctaa tcagaaagcc cacgtcttga cggtacctaa tcagaaagccacggctaact acggctaact acgtgccagc acgtgccage agccgcggta agccgcggta 540 540
atacgtaggt ggcaagcgtt atacgtaggt ggcaagcgttatccggaatt atccggaattattgggcgta attgggcgta aagcgcgcgt aagcgcgcgt aggcggtttt aggcggtttt 600 600
ttaagtctga tgtgaaagcc ttaagtctga tgtgaaagcccacggctcaa cacggctcaaccgtggaggg ccgtggaggg tcattggaaa tcattggaaa ctggaaaact ctggaaaact 660 660
tgagtgcaga agaggaaagt tgagtgcaga agaggaaagtggaattccat ggaattccatgtgtagcggt gtgtagcggt gaaatgcgca gaaatgcgca gagatatgga gagatatgga 720 ggaacaccag tggcgaaggc ggaacaccag tggcgaaggcgactttctgg gactttctggtctgtaactg tctgtaactg acgctgatgt acgctgatgt gcgaaagcgt gcgaaagcgt 780 780 ggggatcaaa caggattaga ggggatcaaa caggattagataccctggta taccctggtagtccacgccg gtccacgccg taaacgatga taaacgatga gtgctaagtg gtgctaagtg 840 840 ttagggggtt tccgcccctt agtgctgcag ttagggggtt tccgcccctt agtgctgcagctaacgcatt ctaacgcatt aagcactccg aagcactccg cctggggagt cctggggagt 900 900 acgaccgcaa ggttgaaact acgaccgcaa ggttgaaactcaaaggaatt caaaggaattgacggggacc gacggggacc cgcacaagcg cgcacaagcg gtggagcatg gtggagcatg 960 960 tggtttaatt cgaagcaacg tggtttaatt cgaagcaacgcgaagaacct cgaagaaccttaccaaatct taccaaatct tgacatcctc tgacatcctc tgacccctct tgacccctct 1020 1020 agagatagag ttttcccctt agagatagag ttttccccttcgggggacag cgggggacagagtgacaggt agtgacaggt ggtgcatggt ggtgcatggt tgtcgtcagc tgtcgtcagc 1080 1080 tcgtgtcgtg agatgttggg tcgtgtcgtg agatgttgggttaagtcccg ttaagtcccgcaacgagcgc caacgagcgc aacccttaag aacccttaag cttagttgcc cttagttgcc 1140 1140 atcattaagt tgggcactct atcattaagt tgggcactctaagttgactg aagttgactgccggtgacaa ccggtgacaa accggaggaa accggaggaa ggtggggatg ggtggggatg 1200 1200 acgtcaaatc atcatgcccc acgtcaaatc atcatgccccttatgatttg ttatgatttgggctacacac ggctacacac gtgctacaat gtgctacaat ggacaataca ggacaataca 1260 1260 aagggcagcg aaaccgcgag aagggcagcg aaaccgcgaggtcaagcaaa gtcaagcaaatcccataaag tcccataaag ttgttctcag ttgttctcag ttcggattgt ttcggattgt 1320 1320 agtctgcaac tcgactatat agtctgcaac tcgactatatgaagctggaa gaagctggaatcgctagtaa tcgctagtaa tcgtagatca tcgtagatca gcatgctacg gcatgctacg 1380 1380 gtgaatacgt tcccgggtct gtgaatacgt tcccgggtcttgtacacacc tgtacacaccgcccgtcaca gcccgtcaca ccacgagagt ccacgagagt ttgtaacacc ttgtaacacc 1440 1440 cgaagccggt ggagtaacca cgaagccggt ggagtaaccatttggagcta tttggagctagccgtcgaag gccgtcgaag gtgggacaaa gtgggacaaa tgattggggt tgattggggt 1500 1500 gaagtcgtaa caaggtagcc gaagtcgtaa caaggtagcc gtatcggaag gtatcggaaggtgcggctgg gtgcggctgg atcacctcct atcacctcct ttct ttct 1554 1554
<210> <210> 44
<211> <211> 1514 1514 <212> <212> DNA DNA <213> <213> Streptococcus pneumoniae Streptococcus pneumoniae
<400> <400> 4 4 gtttgatcct ggctcaggac gtttgatcct ggctcaggacgaacgctggc gaacgctggcggcgtgccta ggcgtgccta atacatgcaa atacatgcaa gtagaacgct gtagaacgct
gaaggaggag cttgcttctc gaaggaggag cttgcttctctggatgagtt tggatgagttgcgaacgggt gcgaacgggt gagtaacgcg gagtaacgcg taggtaacct taggtaacct 120 120
gcctggtagc gggggataac gcctggtagc gggggataactattggaaac tattggaaacgatagctaat gatagctaat accgcataag accgcataag agtggatgtt agtggatgtt 180 180
gcatgacatt tgcttaaaag gcatgacatt tgcttaaaaggtgcacttgc gtgcacttgcatcactacca atcactacca gatggacctg gatggacctg cgttgtatta cgttgtatta 240 240
gctagttggt ggggtaacgg gctagttggt ggggtaacggctcaccaagg ctcaccaaggcgacgataca cgacgataca tagccgacct tagccgacct gagagggtga gagagggtga 300 300
tcggccacac tgggactgag acacggccca tcggccacac tgggactgag acacggcccagactcctacg gactcctacg ggaggcagca ggaggcagca gtagggaatc gtagggaatc 360 360
ttcggcaatg gacggaagtc tgaccgagca ttcggcaatg gacggaagtc tgaccgagcaacgccgcgtg acgccgcgtg agtgaagaag agtgaagaag gttttcggat gttttcggat 420 420
cgtaaagctc tgttgtaaga cgtaaagctc tgttgtaagagaagaacgag gaagaacgagtgtgagagtg tgtgagagtg gaaagttcac gaaagttcac actgtgacgg actgtgacgg 480 480
tatcttacca gaaagggacg tatcttacca gaaagggacggctaactacg gctaactacgtgccagcage tgccagcagc cgcggtaata cgcggtaata cgtaggtccc cgtaggtccc 540 540
gagcgttgtc cggatttatt gagcgttgtc cggatttattgggcgtaaag gggcgtaaagcgagcgcagg cgagcgcagg cggttagata cggttagata agtctgaagt agtctgaagt 600 600
taaaggctgt ggcttaacca taaaggctgt ggcttaaccatagtaggctt tagtaggctttggaaactgt tggaaactgt ttaacttgag ttaacttgag tgcaagaggg tgcaagaggg 660 660
gagagtggaa ttccatgtgt gagagtggaa ttccatgtgtagcggtgaaa agcggtgaaatgcgtagata tgcgtagata tatggaggaa tatggaggaa caccggtggc caccggtggc 720 720
gaaagcggct ctctggcttg gaaagcggct ctctggcttgtaactgacgc taactgacgctgaggctcga tgaggctcga aagcgtgggg aagcgtgggg agcaaacagg agcaaacagg 780 780
attagatacc ctggtagtcc attagatacc ctggtagtccacgctgtaaa acgctgtaaacgatgagtgc cgatgagtgc taggtgttag taggtgttag accctttccg accctttccg 840 gggtttagtg ccgtagctaa gggtttagtg ccgtagctaacgcattaage cgcattaagcactccgcctg actccgcctg gggagtacga gggagtacga ccgcaaggtt ccgcaaggtt 900 900 gaaactcaaa ggaattgacg gaaactcaaa ggaattgacgggggcccgca ggggcccgcacaagcggtgg caagcggtgg agcatgtggt agcatgtggt ttaattcgaa ttaattcgaa 960 960 gcaacgcgaa gaaccttacc gcaacgcgaa gaaccttaccaggtcttgac aggtcttgacatccctctga atccctctga ccgctctaga ccgctctaga gatagagttt gatagagttt 1020 1020 tccttcggga cagaggtgac tccttcggga cagaggtgacaggtggtgca aggtggtgcatggttgtcgt tggttgtcgt cagctcgtgt cagctcgtgt cgtgagatgt cgtgagatgt 1080 1080 tgggttaagt cccgcaacga tgggttaagt cccgcaacgagcgcaacccc gcgcaacccctattgttagt tattgttagt tgccatcatt tgccatcatt cagttgggca cagttgggca 1140 1140 ctctagcgag actgccggta ctctagcgag actgccggtaataaaccgga ataaaccggaggaaggtggg ggaaggtggg gatgacgtca gatgacgtca aatcatcatg aatcatcatg 1200 1200 ccccttatga cctgggctac ccccttatga cctgggctacacacgtgcta acacgtgctacaatggctgg caatggctgg tacaacgagt tacaacgagt cgcaagccgg cgcaagccgg 1260 1260 tgacggcaag ctaatctctt tgacggcaag ctaatctcttaaagccagtc aaagccagtctcagttcgga tcagttcgga ttgtaggctg ttgtaggctg caactcgcct caactcgcct 1320 1320 acatgaagtc ggaatcgcta acatgaagtc ggaatcgctagtaatcgcgg gtaatcgcggatcagcacgc atcagcacgc cgcggtgaat cgcggtgaat acgttcccgg acgttcccgg 1380 1380 gccttgtaca caccgcccgt gccttgtaca caccgcccgtcacaccacga cacaccacgagagtttgtaa gagtttgtaa cacccgaagt cacccgaagt cggtgaggta cggtgaggta 1440 1440 accgtaagga gccagccgcc accgtaagga gccagccgcctaaggtggga taaggtgggatagatgattg tagatgattg gggtgaagtc gggtgaagtc gtaacaaggt gtaacaaggt 1500 1500 a g c c g t a t c g g a a g a g C C g t a t C g 1514 1514 g a a g
<210> <210> 5 5 <211> <211> 1501 1501 <212> <212> DNA DNA <213> <213> Streptococcusagalactiae Streptococcus agalactiae
<400> <400> 5 5 gacgaacgct ggcggcgtgcctaatacatg gacgaacgct ggcggcgtgc ctaatacatgcaagtagaac caagtagaac gctgaggttt gctgaggttt ggtgtttaca ggtgtttaca
ctagactgat gagttgcgaacgggtgagta ctagactgat gagttgcgaa cgggtgagtaacgcgtaggt acgcgtaggt aacctgcctc aacctgcctc atagcggggg atagcggggg 120 120 ataactattg gaaacgatag ataactattg gaaacgatagctaataccgc ctaataccgcataagagtaa ataagagtaa ttaacacatg ttaacacatg ttagttattt ttagttattt 180 180 aaaaggagca attgcttcac aaaaggagca attgcttcactgtgagatgg tgtgagatggacctgcgttg acctgcgttg tattagctag tattagctag ttggtgaggt ttggtgaggt 240 240 aaaggctcac caaggcgacg aaaggctcac caaggcgacgatacatagcc atacatagccgacctgagag gacctgagag ggtgatcggc ggtgatcggc cacactggga cacactggga 300 300 ctgagacacg gcccagactc ctgagacacg gcccagactcctacgggagg ctacgggaggcagcagtagg cagcagtagg gaatcttcgg gaatcttcgg caatggacgg caatggacgg 360 360 aagtctgacc gagcaacgcc aagtctgacc gagcaacgccgcgtgagtga gcgtgagtgaagaaggtttt agaaggtttt cggatcgtaa cggatcgtaa agctctgttg agctctgttg 420 420 ttagagaaga acgttggtag ttagagaaga acgttggtaggagtggaaaa gagtggaaaatctaccaagt tctaccaagt gacggtaact gacggtaact aaccagaaag aaccagaaag 480 480 ggacggctaa ctacgtgcca ggacggctaa ctacgtgccagcagccgcgg gcagccgcggtaatacgtag taatacgtag gtcccgagcg gtcccgagcg ttgtccggat ttgtccggat 540 540 ttattgggcg taaagcgagcgcaggcggtt ttattgggcg taaagcgagc gcaggcggttctttaagtct ctttaagtct gaagttaaag gaagttaaag gcagtggctt gcagtggctt 600 600 aaccattgta cgctttggaa aaccattgta cgctttggaaactggaggac actggaggacttgagtgcag ttgagtgcag aaggggagag aaggggagag tggaattcca tggaattcca 660 660 tgtgtagcgg tgaaatgcgt tgtgtagcgg tgaaatgcgtagatatatgg agatatatggaggaacaccg aggaacaccg gtggcgaaag gtggcgaaag cggctctctg cggctctctg 720 720 gtctgtaact gacgctgagg gtctgtaact gacgctgaggctcgaaagcg ctcgaaagcgtggggagcaa tggggagcaa acaggattag acaggattag ataccctggt ataccctggt 780 780 agtccacgcc gtaaacgatg agtccacgcc gtaaacgatgagtgctaggt agtgctaggtgttaggccct gttaggccct ttccggggct ttccggggct tagtgccgca tagtgccgca 840 840 gctaacgcat taagcactcc gctaacgcat taagcactccgcctggggag gcctggggagtacgaccgca tacgaccgca aggttgaaac aggttgaaac tcaaaggaat tcaaaggaat 900 900 tgacgggggc ccgcacaage tgacgggggc ccgcacaagcggtggagcat ggtggagcatgtggtttaat gtggtttaat tcgaagcaac tcgaagcaac gcgaagaacc gcgaagaacc 960 ttaccaggtc ttgacatcct tctgaccggc ttaccaggtc ttgacatcct tctgaccggcctagagatag ctagagatag gctttctctt gctttctctt cggagcagaa cggagcagaa 1020 1020 gtgacaggtg gtgcatggtt gtgacaggtg gtgcatggttgtcgtcagct gtcgtcagctcgtgtcgtga cgtgtcgtga gatgttgggt gatgttgggt taagtcccgc taagtcccgc 1080 1080 aacgagcgca acccctattg aacgagcgca acccctattgttagttgcca ttagttgccatcattaagtt tcattaagtt gggcactcta gggcactcta gcgagactgc gcgagactgc 1140 1140 cggtaataaa ccggaggaag cggtaataaa ccggaggaaggtggggatga gtggggatgacgtcaaatca cgtcaaatca tcatgcccct tcatgcccct tatgacctgg tatgacctgg 1200 1200 gctacacacg tgctacaatg gctacacacg tgctacaatggttggtacaa gttggtacaacgagtcgcaa cgagtcgcaa gccggtgacg gccggtgacg gcaagctaat gcaagctaat 1260 1260 ctcttaaagc caatctcagt ctcttaaage caatctcagttcggattgta tcggattgtaggctgcaact ggctgcaact cgcctacatg cgcctacatg aagtcggaat aagtcggaat 1320 1320 cgctagtaat cgcggatcag cgctagtaat cgcggatcagcacgccgcgg cacgccgcggtgaatacgtt tgaatacgtt cccgggcctt cccgggcctt gtacacaccg gtacacaccg 1380 1380 cccgtcacac cacgagagtt cccgtcacac cacgagagtttgtaacaccc tgtaacacccgaagtcggtg gaagtcggtg aggtaacctt aggtaacctt ttaggagcca ttaggagcca 1440 1440 gccgcctaag gtgggataga gccgcctaag gtgggatagatgattggggt tgattggggtgaagtcgtaa gaagtcgtaa caaggtagcc caaggtagcc gtatcggaag gtatcggaag 1500 1500 g g 1501 1501
<210> <210> 6 6 <211> <211> 1543 1543 <212> <212> DNA DNA <213> <213> Streptococcuspyogenes Streptococcus pyogenes
<400> <400> 66 gagagtttga tcctggctcaggacgaacgc gagagtttga tcctggctca ggacgaacgctggcggcgtg tggcggcgtg cctaatacat cctaatacat gcaagtagaa gcaagtagaa
cgctgagaac tggtgcttgc cgctgagaac tggtgcttgcaccggttcaa accggttcaaggagttgcga ggagttgcga acgggtgagt acgggtgagt aacgcgtagg aacgcgtagg 120 120
taacctacct catagcgggg taacctacct catagcgggggataactatt gataactattggaaacgata ggaaacgata gctaataccg gctaataccg cataagagag cataagagag 180 actaacgcat gttagtaatt actaacgcat gttagtaatttaaaaggggc taaaaggggcaattgctcca aattgctcca ctatgagatg ctatgagatg gacctgcgtt gacctgcgtt 240 240 gtattagcta gttggtgagg gtattagcta gttggtgaggtaaaggctca taaaggctcaccaaggcgac ccaaggcgac gatacatagc gatacatage cgacctgaga cgacctgaga 300 300 gggtgatcgg ccacactggg gggtgatcgg ccacactgggactgagacac actgagacacggcccagact ggcccagact cctacgggag cctacgggag gcagcagtag gcagcagtag 360 360 ggaatcttcg gcaatggggg ggaatcttcg gcaatgggggcaaccctgac caaccctgaccgagcaacgc cgagcaacgc cgcgtgagtg cgcgtgagtg aagaaggttt aagaaggttt 420 420 tcggatcgta aagctctgtt tcggatcgta aagctctgttgttagagaag gttagagaagaatgatggtg aatgatggtg ggagtggaaa ggagtggaaa atccaccaag atccaccaag 480 480 tgacggtaac taaccagaaagggacggcta tgacggtaac taaccagaaa gggacggctaactacgtgcc actacgtgcc agcagccgcg agcagccgcg gtaatacgta gtaatacgta 540 540 ggtcccgagc gttgtccgga ggtcccgagc gttgtccggatttattgggc tttattgggcgtaaagcgag gtaaagcgag cgcaggcggt cgcaggcggt tttttaagtc tttttaagtc 600 600 tgaagttaaa ggcattggct tgaagttaaa ggcattggctcaaccaatgt caaccaatgtacgctttgga acgctttgga aactggagaa aactggagaa cttgagtgca cttgagtgca 660 660 gaaggggaga gtggaattcc gaaggggaga gtggaattccatgtgtagcg atgtgtagcggtgaaatgcg gtgaaatgcg tagatatatg tagatatatg gaggaacacc gaggaacacc 720 720 ggtggcgaaa gcggctctct ggtggcgaaa gcggctctctggtctgtaac ggtctgtaactgacgctgag tgacgctgag gctcgaaagc gctcgaaaga gtggggagca gtggggagca 780 780 aacaggatta gataccctgg aacaggatta gataccctggtagtccacgc tagtccacgccgtaaacgat cgtaaacgat gagtgctagg gagtgctagg tgttaggccc tgttaggccc 840 840 tttccggggc ttagtgccggagctaacgca tttccggggc ttagtgccgg agctaacgcattaagcactc ttaagcactc cgcctgggga cgcctgggga gtacgaccgc gtacgaccgc 900 900 aaggttgaaa ctcaaaggaa aaggttgaaa ctcaaaggaattgacggggg ttgacgggggcccgcacaag cccgcacaag cggtggagca cggtggagca tgtggtttaa tgtggtttaa 960 960 ttcgaagcaa cgcgaagaac ttcgaagcaa cgcgaagaaccttaccaggt cttaccaggtcttgacatcc cttgacatcc cgatgcccgc cgatgcccgc tctagagata tctagagata 1020 1020 gagttttact tcggtacatc gagttttact tcggtacatcggtgacaggt ggtgacaggtggtgcatggt ggtgcatggt tgtcgtcagc tgtcgtcagc tcgtgtcgtg tcgtgtcgtg 1080 agatgttggg ttaagtcccg agatgttggg ttaagtcccgcaacgagcgc caacgagcgcaacccctatt aacccctatt gttagttgcc gttagttgcc atcattaagt atcattaagt 1140 1140 tgggcactct agcgagactg tgggcactct agcgagactgccggtaataa ccggtaataaaccggaggaa accggaggaa ggtggggatg ggtggggatg acgtcaaatc acgtcaaatc 1200 1200 atcatgcccc ttatgacctg atcatgcccc ttatgacctgggctacacac ggctacacacgtgctacaat gtgctacaat ggttggtaca ggttggtaca acgagtcgca acgagtcgca 1260 1260 agccggtgac ggcaagctaa agccggtgac ggcaagctaatctcttaaag tctcttaaagccaatctcag ccaatctcag ttcggattgt ttcggattgt aggctgcaac aggctgcaac 1320 1320 tcgcctacat gaagtcggaa tcgcctacat gaagtcggaatcgctagtaa tcgctagtaatcgcggatca tcgcggatca gcacgccgcg gcacgccgcg gtgaatacgt gtgaatacgt 1380 1380 tcccgggcct tgtacacacc tcccgggcct tgtacacaccgcccgtcaca gcccgtcacaccacgagagt ccacgagagt ttgtaacacc ttgtaacacc cgaagtcggt cgaagtcggt 1440 1440 gaggtaacct attaggagec gaggtaacct attaggagccagccgcctaa agccgcctaaggtgggatag ggtgggatag atgattgggg atgattgggg tgaagtcgta tgaagtcgta 1500 1500 acaaggtagc cgtatcggaa ggtgcggctg gatcacctcc ttt acaaggtage 1543 1543 cgtatcggaa ggtgcggctg gatcacctcc ttt
<210> <210> 7 7 <211> <211> 1522 1522 <212> <212> DNA DNA <213> <213> Enterococcusfaecalis Enterococcus faecalis
<400> <400> 77 agagtttgat cctggctcag agagtttgat cctggctcaggacgaacgct gacgaacgctggcggcgtgc ggcggcgtgc ctaatacatg ctaatacatg caagtcgaac caagtcgaac
gcttctttcc tcccgagtgc gcttctttcc tcccgagtgcttgcactcaa ttgcactcaattggaaagag ttggaaagag gagtggcgga gagtggcgga cgggtgagta cgggtgagta 120 120
acacgtgggt aacctaccca acacgtgggt aacctacccatcagaggggg tcagagggggataacacttg ataacacttg gaaacaggtg gaaacaggtg ctaataccgc ctaataccgc 180 180
ataacagttt atgccgcatg ataacagttt atgccgcatggcataagagt gcataagagtgaaaggcgct gaaaggcgct ttcgggtgtc ttcgggtgtc gttgatggat gttgatggat 240 240
ggacccgcgg tgcattagct ggacccgcgg tgcattagctagttggtgag agttggtgaggtaacggctc gtaacggctc accaaggcca accaaggcca cgatgcatag cgatgcatag 300 ccgacctgag agggtgatcg ccgacctgag agggtgatcggccacactgg gccacactgggactgagaca gactgagaca cggcccagac cggcccagac tcctacggga tcctacggga 360 360 ggcagcagta gggaatcttc ggcagcagta gggaatcttcggcaatagac ggcaatggacgaaagtctga gaaagtctga ccgagcaacg ccgagcaacg ccgcgtgagt ccgcgtgagt 420 420 gaagaaggtt ttcggatcgt gaagaaggtt ttcggatcgtaaaactctgt aaaactctgttgttagagaa tgttagagaa gaacaaggac gaacaaggac gttagtaact gttagtaact 480 480 gaacgtcccc tgacggtatc gaacgtcccc tgacggtatctaaccagaaa taaccagaaagccacggcta gccacggcta actacgtgcc actacgtgcc agcagccgcg agcagccgcg 540 540 gtaatacgta ggtggcaage gtaatacgta ggtggcaagcgttgtccgga gttgtccggatttattgggc tttattgggc gtaaagcgag gtaaagcgag cgcaggcggt cgcaggcggt 600 600 ttcttaagtc tgatgtgaaa gcccccggct ttcttaagtc tgatgtgaaa gcccccggctcaaccgggga caaccgggga gggtcattgg gggtcattgg aaactgggag aaactgggag 660 660 acttgagtgc agaagaggag acttgagtgc agaagaggagagtggaattc agtggaattccatgtgtagc catgtgtagc ggtgaaatgc ggtgaaatgc gtagatatat gtagatatat 720 720 ggaggaacac cagtggcgaa ggaggaacac cagtggcgaaggcggctctc ggcggctctctggtctgtaa tggtctgtaa ctgacgctga ctgacgctga ggctcgaaag ggctcgaaag 780 780 cgtggggagc aaacaggatt cgtggggagc aaacaggattagataccctg agataccctggtagtccacg gtagtccacg ccgtaaacga ccgtaaacga tgagtgctaa tgagtgctaa 840 840 gtgttggagg gtttccgccc gtgttggagg gtttccgcccttcagtgctg ttcagtgctgcagcaaacgc cagcaaacgc attaagcact attaagcact ccgcctgggg ccgcctgggg 900 900 agtacgaccg caaggttgaa agtacgaccg caaggttgaaactcaaagga actcaaaggaattgacgggg attgacgggg gcccgcacaa gcccgcacaa gcggtggagc gcggtggagc 960 960 atgtggttta attcgaagca atgtggttta attcgaagcaacgcgaagaa acgcgaagaaccttaccagg ccttaccagg tcttgacatc tcttgacatc ctttgaccac ctttgaccac 1020 1020 tctagagata gagctttccc tctagagata gagctttcccttcggggaca ttcggggacaaagtgacagg aagtgacagg tggtgcatgg tggtgcatgg ttgtcgtcag ttgtcgtcag 1080 1080 ctcgtgtcgt gagatgttgg ctcgtgtcgt gagatgttgggttaagtccc gttaagtcccgcaacgagcg gcaacgagcg caacccttat caacccttat tgttagttgc tgttagttgc 1140 1140 catcatttag ttgggcactctagcgagact catcatttag ttgggcactc tagcgagactgccggtgaca gccggtgaca aaccggagga aaccggagga aggtggggat aggtggggat 1200 gacgtcaaat catcatgccc gacgtcaaat catcatgccccttatgacct cttatgacctgggctacaca gggctacaca cgtgctacaa cgtgctacaa tgggaagtac tgggaagtac 1260 1260 aacgagtcgc tagaccgcga ggtcatgcaa aacgagtcgc tagaccgcga ggtcatgcaaatctcttaaa atctcttaaa gcttctctca gcttctctca gttcggattg gttcggattg 1320 1320 caggctgcaa ctcgcctgca caggctgcaa ctcgcctgcatgaagccgga tgaagccggaatcgctagta atcgctagta atcgcggatc atcgcggatc agcacgccgc agcacgccgc 1380 1380 ggtgaatacg ttcccgggcc ggtgaatacg ttcccgggccttgtacacac ttgtacacaccgcccgtcac cgcccgtcac accacgagag accacgagag tttgtaacac tttgtaacac 1440 1440 ccgaagtcgg tgaggtaacctttttggage ccgaagtcgg tgaggtaacc tttttggagccagccgccta cagccgccta aggtgggata aggtgggata gatgattggg gatgattggg 1500 1500 g t g a a g t c g t a a c a a g g t a g c c gtgaagtcgt 1522 1522 aacaaggtag cc
<210> <210> 8 8 <211> <211> 1551 1551 <212> <212> DNA DNA <213> <213> Enterococcus faecium Enterococcus faecium
<400> <400> 88 agagtttgat cctggctcaggacgaacgct agagtttgat cctggctcag gacgaacgctggcggcgtgc ggcggcgtgc ctatacatgc ctatacatgc aagtcgaacg aagtcgaacg
cttctttttc caccggagct tgctccaccg cttctttttc caccggagct tgctccaccggaaaaagagg gaaaaagagg agtggcgaac agtggcgaac gggtgagtaa gggtgagtaa 120 120
cacgtgggta acctgcccat cacgtgggta acctgcccatcagaaaggga cagaaagggataacacttgg taacacttgg aaacaggtgc aaacaggtgc taataccgta taataccgta 180 180
taacaaatca aaaccgcatggttttgattt taacaaatca aaaccgcatg gttttgatttgaaaggcgct gaaaggcgct ttcgggtgtc ttcgggtgtc gctgatggat gctgatggat 240 240
ggacccgcgg tgcattagct ggacccgcgg tgcattagctagttggtgag agttggtgaggtaacggctc gtaacggctc accaaggcca accaaggcca cgatgcatag cgatgcatag 300 300
ccgcacctga gagggtgatc ccgcacctga gagggtgatcggccacattg ggccacattgggactgagac ggactgagac acggcccaaa acggcccaaa ctctacggga ctctacggga 360 360
ggcagcagta gggaatcttc ggcagcagta gggaatcttcggcaatggac ggcaatggacgaaagtctga gaaagtctga ccgagcaacg ccgagcaacg ccgcgtgagt ccgcgtgagt 420 gaagaaggtt ttcggatcgt gaagaaggtt ttcggatcgtaaaactctgt aaaactctgttgttagagaa tgttagagaa gaacaaggat gaacaaggat gagagtaact gagagtaact 480 480 gttcatccct tgacggtatc gttcatccct tgacggtatctaaccagaaa taaccagaaagccacggcta gccacggcta actacgtgcc actacgtgcc agcagccgcg agcagccgcg 540 540 gtaatacgta ggtggcaagc gtaatacgta ggtggcaagcgttgtccgga gttgtccggatttattgggc tttattgggc gtaaagcgag gtaaagcgag cgcaggcggt cgcaggcggt 600 600 tcttaagtct gatgtgaaag tcttaagtct gatgtgaaagcccccggctc cccccggctcaaccggggag aaccggggag ggtcattgga ggtcattgga aactgggaga aactgggaga 660 660 cttgagtgca gaagaggaga cttgagtgca gaagaggagagtggaattcc gtggaattccatgtgtagcg atgtgtagcg gtgaaatgcg gtgaaatgcg tagatatatg tagatatatg 720 720 gaggaacacc agtggcgaag gaggaacacc agtggcgaaggcggctctct gcggctctctggtctgtaac ggtctgtaac tgacgctgag tgacgctgag gctcgaaagc gctcgaaagc 780 780 gtggggagca aacaggatta gtggggagca aacaggattagataccctgg gataccctggtagtccacgc tagtccacgc cgtaaacgat cgtaaacgat gagtgctaag gagtgctaag 840 840 tgttggaggg tttccgccct tgttggaggg tttccgcccttcagtgctgc tcagtgctgcagctaacgca agctaacgca ttaagcactc ttaagcactc cgcctgggga cgcctgggga 900 900 gtacgaccgc aaggttgaaa gtacgaccgc aaggttgaaactcaaaggaa ctcaaaggaattgacggggg ttgacggggg cccgcacaag cccgcacaag cggtggagca cggtggagca 960 960 tgtggtttaa ttcgaagcaa tgtggtttaa ttcgaagcaacacgaagaac cacgaagaaccttaccaggt cttaccaggt cttgacatcc cttgacatcc tttgaccact tttgaccact 1020 1020 ctagagatag agcttcccct tcgggggcaa ctagagatag agcttcccct tcgggggcaaagtgacaggt agtgacaggt ggtgcatggt ggtgcatggt tgtcgtcagc tgtcgtcagc 1080 1080 tcgtgtcgtg agatgttgggttaagtcccg tcgtgtcgtg agatgttggg ttaagtcccgcaacgagcgc caacgagcgc aacccttatt aacccttatt gttagttgcc gttagttgcc 1140 1140 atcattcagt tgggcactct atcattcagt tgggcactctagcaagactg agcaagactgccggtgacaa ccggtgacaa accggaggaa accggaggaa ggtggggatg ggtggggatg 1200 1200 acgtcaaatc atcatgcccc acgtcaaatc atcatgccccttatgacctg ttatgacctgggctacacac ggctacacac gtgctacaat gtgctacaat gggaagtaca gggaagtaca 1260 1260 acgagttgcg aagtcgcgag acgagttgcg aagtcgcgaggctaagctaa gctaagctaatctcttaaag tctcttaaag cttctctcag cttctctcag ttcggattgc ttcggattgc 1320 aggctgcaac tcgcctgcat aggctgcaac tcgcctgcatgaagccggaa gaagccggaatcgctagtaa tcgctagtaa tcgcggatca tcgcggatca gcacgccgcg gcacgccgcg 1380 1380 tgaatacgtt cccgggcctt tgaatacgtt cccgggccttgtacacaccg gtacacaccgcccgtcacac cccgtcacac cacgagagtt cacgagagtt tgtaacaccc tgtaacaccc 1440 1440 gaagtcggtg aggtaacctt gaagtcggtg aggtaaccttttggagccag ttggagccagccgcctaagg ccgcctaagg tgggatagat tgggatagat gattggggtg gattggggtg 1500 1500 aagtcgtaac aaggtagccg tatctgaagg tgcggctgga tcacctcctt t t aagtcgtaac aaggtagccg tatctgaagg tgcggctgga tcacctcctt 1551 1551
<210> <210> 9 9 <211> <211> 1542 1542 <212> <212> DNA DNA <213> <213> Proteus mirabilis Proteus mirabilis
<400> 99 <400> aattgaagag tttgatcatg aattgaagag tttgatcatggctcagattg gctcagattgaacgctggcg aacgctggcg gcaggcctaa gcaggcctaa cacatgcaag cacatgcaag
tcgagcggta acaggagaaa tcgagcggta acaggagaaagcttgctttc gcttgctttcttgctgacga ttgctgacga gcggcggacg gcggcggacg ggtgagtaat ggtgagtaat 120 120
gtatggggat ctgcccgata gtatggggat ctgcccgatagagggggata gagggggataactactggaa actactggaa acggtggcta acggtggcta ataccgcata ataccgcata 180 180
atgtctacgg accaaagcag atgtctacgg accaaagcaggggctcttcg gggctcttcggaccttgcac gaccttgcac tatcggatga tatcggatga acccatatgg acccatatgg 240 240
gattagctag taggtggggt gattagctag taggtggggtaaaggctcac aaaggctcacctaggcgacg ctaggcgacg atctctagct atctctagct ggtctgagag ggtctgagag 300 300
gatgatcagc cacactggga gatgatcage cacactgggactgagacacg ctgagacacggcccagacto gcccagactc ctacgggagg ctacgggagg cagcagtggg cagcagtggg 360 360
gaatattgca caatgggcgc gaatattgca caatgggcgcaagcctgatg aagcctgatgcagccatgcc cagccatgcc gcgtgtatga gcgtgtatga agaaggcctt agaaggcctt 420 420
agggttgtaa agtactttca agggttgtaa agtactttcagcggggagga gcggggaggaaggtgataag aggtgataag gttaataccc gttaataccc ttgtcaattg ttgtcaattg 480 480
acgttacccg cagaagaage acgttacccg cagaagaagcaccggctaac accggctaactccgtgccag tccgtgccag cagccgcggt cagccgcggt aatacggagg aatacggagg 540 gtgcaagcgt taatcggaat gtgcaagcgt taatcggaattactgggcgt tactgggcgtaaagcgcacg aaagcgcacg caggcggtca caggcggtca attaagtcag attaagtcag 600 600 atgtgaaagc cccgagctta atgtgaaago cccgagcttaacttgggaat acttgggaattgcatctgaa tgcatctgaa actggttggc actggttggc tagagtcttg tagagtcttg 660 660 tagagggggg tagaattcca tgtgtagcgg tagagggggg tagaattcca tgtgtagcggtgaaatgcgt tgaaatgcgt agagatgtgg agagatgtgg aggaataccg aggaataccg 720 720 gtggcgaagg cggccccctg gtggcgaagg cggccccctggacaaagact gacaaagactgacgctcagg gacgctcagg tgcgaaagcg tgcgaaagcg tggggagcaa tggggagcaa 780 780 acaggattag ataccctggt acaggattag ataccctggtagtccacgct agtccacgctgtaaacgatg gtaaacgatg tcgatttaga tcgatttaga ggttgtggtc ggttgtggtc 840 840 ttgaaccgtg gcttctggag ctaacgcgtt ttgaaccgtg gcttctggag ctaacgcgttaaatcgaccg aaatcgaccg cctggggagt cctggggagt acggccgcaa acggccgcaa 900 900 ggttaaaact caaatgaatt ggttaaaact caaatgaattgacgggggcc gacgggggcccgcacaagcg cgcacaagcg gtggagcatg gtggagcatg tggtttaatt tggtttaatt 960 960 cgatgcaacg cgaagaacct cgatgcaacg cgaagaaccttacctactct tacctactcttgacatccag tgacatccag cgaatccttt cgaatccttt agagatagag agagatagag 1020 1020 gagtgccttc gggaacgctg gagtgccttc gggaacgctgagacaggtgc agacaggtgctgcatggctg tgcatggctg tcgtcagctc tcgtcagctc gtgttgtgaa gtgttgtgaa 1080 1080 atgttgggtt aagtcccgca atgttgggtt aagtcccgcaacgagcgcaa acgagcgcaacccttatcct cccttatcct ttgttgccag ttgttgccag cacgtaatgg cacgtaatgg 1140 1140 tgggaactca aaggagactg tgggaactca aaggagactgccggtgataa ccggtgataaaccggaggaa accggaggaa ggtggggatg ggtggggatg acgtcaagtc acgtcaagtc 1200 1200 atcatggccc ttacgagtag atcatggccc ttacgagtagggctacacac ggctacacacgtgctacaat gtgctacaat ggcagataca ggcagataca aagagaagcg aagagaagcg 1260 1260 acctcgcgag agcaagcgga acctcgcgag agcaagcggaactcataaag actcataaagtctgtcgtag tctgtcgtag tccggattgg tccggattgg agtctgcaac agtctgcaac 1320 1320 tcgactccat gaagtcggaa tcgactccat gaagtcggaatcgctagtaa tcgctagtaatcgtagatca tcgtagatca gaatgctacg gaatgctacg gtgaatacgt gtgaatacgt 1380 1380 tcccgggcct tgtacacacc tcccgggcct tgtacacaccgcccgtcaca gcccgtcacaccatgggagt ccatgggagt gggttgcaaa gggttgcaaa agaagtaggt agaagtaggt 1440 agcttaacct tcgggagggc agcttaacct tcgggagggcgcttaccact gcttaccactttgtgattca ttgtgattca tgactggggt tgactggggt gaagtcgtaa gaagtcgtaa 1500 1500 caaggtaacc gtaggggaac ctgcggttgg atcacctcct ta caaggtaaco 1542 1542 gtaggggaac ctgcggttgg atcacctcct ta
<210> <210> 10 10 <211> <211> 1505 1505 <212> <212> DNA DNA <213> <213> Serratia marcescens Serratia marcescens
<400> <400> 10 10 gctcagattg aacgctggcggcaggcttaa gctcagattg aacgctggcg gcaggcttaacacatgcaag cacatgcaag tcgagcggta tcgagcggta gcacagggga gcacagggga
gcttgctccc tgggtgacga gcttgctccc tgggtgacgagcggcggacg gcggcggacgggtgagtaat ggtgagtaat gtctgggaaa gtctgggaaa ctgcctgatg ctgcctgatg 120 120
gagggggata actactggaa gagggggata actactggaaacggtagcta acggtagctaataccgcata ataccgcata acgtcgcaag acgtcgcaag accaaagagg accaaagagg 180 180
gggaccttcg ggcctcttgc gggaccttcg ggcctcttgccatcagatgt catcagatgtgcccagatgg gcccagatgg gattagctag gattagctag taggtggggt taggtggggt 240 240
aatggctcac ctaggcgacg aatggctcac ctaggcgacgatccctagct atccctagctggtctgagag ggtctgagag gatgaccagc gatgaccage cacactggaa cacactggaa 300 300
ctgagacacg gtccagactc ctgagacacg gtccagactcctacgggagg ctacgggaggcagcagtggg cagcagtggg gaatattgca gaatattgca caatgggcgc caatgggcgc 360 360
aagcctgatg cagccatgcc aagcctgatg cagccatgccgcgtgtgtga gcgtgtgtgaagaaggcctt agaaggcctt cgggttgtaa cgggttgtaa agcactttca agcactttca 420 420
gcgaggagga aggtggtgag gcgaggagga aggtggtgagcttaatacgt cttaatacgttcatcaattg tcatcaattg acgttactcg acgttactcg cagaagaagc cagaagaage 480 480
accggctaac tccgtgccag accggctaac tccgtgccagcagccgcggt cagccgcggtaatacggagg aatacggagg gtgcaagcgt gtgcaagcgt taatcggaat taatcggaat 540 540
tactgggcgt aaagcgcacgcaggcggttt tactgggcgt aaagcgcacg caggcggtttgttaagtcag gttaagtcag atgtgaaatc atgtgaaatc cccgggctca cccgggctca 600 600
acctgggaac tgcatttgaa acctgggaac tgcatttgaaactggcaage actggcaagctagagtctcg tagagtctcg tagagggggg tagagggggg tagaattcca tagaattcca 660 ggtgtagcgg tgaaatgcgt ggtgtagcgg tgaaatgcgtagagatctgg agagatctggaggaataccg aggaataccg gtggcgaagg gtggcgaagg cgggcccctg cgggcccctg 720 720 gacgaagact gacgctcagg gacgaagact gacgctcaggtgccaaagcg tgccaaagcgtggggagcaa tggggagcaa acaggattag acaggattag ataccctggt ataccctggt 780 780 agtccacgct gtaaacgatg agtccacgct gtaaacgatgtcgatttgga tcgatttggaggttgtgccc ggttgtgccc ttgaggcgtg ttgaggcgtg gcttccggag gcttccggag 840 840 ctaacgcgtt aaatcgaccg ctaacgcgtt aaatcgaccgcctggggagt cctggggagtacggccgcaa acggccgcaa ggttaaaact ggttaaaact caaatgaatt caaatgaatt 900 900 gacgggggcc cgcacaagcg gacgggggcc cgcacaagcggtggagcatg gtggagcatgtggtttaatt tggtttaatt cgatgcaacg cgatgcaacg cgaagaacct cgaagaacct 960 960 tacctactct tgacatccag tacctactct tgacatccagagaactttcc agaactttccagagatggat agagatggat tggtgccttc tggtgccttc gggaactctg gggaactctg 1020 1020 agacaggtgc tgcatggctg agacaggtgc tgcatggctgtcgtcagctc tcgtcagctcgtgttgtgaa gtgttgtgaa atgttgggtt atgttgggtt aagtcccgca aagtcccgca 1080 1080 acgagcgcaa cccttatcct acgagcgcaa cccttatcctttgttgccag ttgttgccagcggttcggcc cggttcggcc gggaactcaa gggaactcaa aggagactgc aggagactgc 1140 1140 cagtgataaa ctggaggaag cagtgataaa ctggaggaaggtggggatga gtggggatgacgtcaagtca cgtcaagtca tcatggccct tcatggccct tacgagtagg tacgagtagg 1200 1200 gctacacacg tgctacaatg gctacacacg tgctacaatggcatatacaa gcatatacaaagagaagcga agagaagcga cctcgcgaga cctcgcgaga gcaagcggac gcaagcggac 1260 1260 ctcataaagt atgtcgtagt ctcataaagt atgtcgtagtccggattgga ccggattggagtctgcaact gtctgcaact cgactccatg cgactccatg aagtcggaat aagtcggaat 1320 1320 cgctagtaat cgtagatcag cgctagtaat cgtagatcagaatgctacgg aatgctacggtgaatacgtt tgaatacgtt cccgggcctt cccgggcctt gtacacaccg gtacacaccg 1380 1380 cccgtcacac catgggagtg cccgtcacac catgggagtgggttgcaaaa ggttgcaaaagaagtaggta gaagtaggta gcttaacctt gcttaacctt cgggagggcg cgggagggcg 1440 1440 cttaccactt tgtgattcatgactggggtg cttaccactt tgtgattcat gactggggtgaagtcgtaac aagtcgtaac aaggtaaccg aaggtaaccg taggggaacc taggggaacc 1500 1500 t t g g c C g g g g 1505
<210> <210> 11 11 <211> <211> 1438 1438 <212> <212> DNA DNA <213> <213> Enterobacteraerogenes Enterobacter aerogenes
<220> <220> <221> misc_feature <221> misc_feature <222> (1425)..(1425) <222> (1425)..(1425) <223> n isa, <223> n is a,C, c,g,g,or ortt
<400> <400> 11 11 acgctggcgg caggcctaacacatgcaagt acgctggcgg caggectaac acatgcaagtcgagcggtag cgagcggtag cacagagagc cacagagage ttgctctcgg ttgctctcgg
gtgacgagcg gcggacgggt gtgacgagcg gcggacgggtgagtaatgtc gagtaatgtctgggaaactg tgggaaactg cctgatggag cctgatggag ggggataact ggggataact 120 120
actggaaacg gtagctaata actggaaacg gtagctaataccgcataacg ccgcataacgtcgcaagacc tcgcaagacc aaagtggggg aaagtggggg accttcgggc accttcgggc 180 180
ctcatgccat cagatgtgcc ctcatgccat cagatgtgcccagatgggat cagatgggattagctagtag tagctagtag gtggggtaat gtggggtaat ggctcaccta ggctcaccta 240 240
ggcgacgatc cctagctggt ggcgacgatc cctagctggtctgagaggat ctgagaggatgaccagccac gaccagccac actggaactg actggaactg agacacggtc agacacggtc 300 300
cagactccta cgggaggcag cagactecta cgggaggcagcagtggggaa cagtggggaatattgcacaa tattgcacaa tgggcgcaag tgggcgcaag cctgatgcag cctgatgcag 360 360
ccatgccgcg tgtatgaaga ccatgccgcg tgtatgaagaaggccttcgg aggccttcgggttgtaaagt gttgtaaagt actttcagcg actttcagcg aggaggaagg aggaggaagg 420 420
cgttaaggtt aataaccttg cgttaaggtt aataaccttggcgattgacg gcgattgacgttactcgcag ttactcgcag aagaagcacc aagaagcacc ggctaactcc ggctaactcc 480 480
gtgccagcag ccgcggtaat gtgccagcag ccgcggtaatacggagggtg acggagggtgcaagcgttaa caagcgttaa tcggaattac tcggaattac tgggcgtaaa tgggcgtaaa 540 540
gcgcacgcag gcggtctgtc gcgcacgcag gcggtctgtcaagtcggatg aagtcggatgtgaaatcccc tgaaatcccc gggctcaacc gggctcaacc tgggaactgc tgggaactgc 600 600
attcgaaact ggcaggctag attcgaaact ggcaggctagagtcttgtag agtcttgtagaggggggtag aggggggtag aattccaggt aattccaggt gtagcggtga gtagcggtga 660 aatgcgtaga gatctggagg aatgcgtaga gatctggaggaataccggtg aataccggtggcgaaggcgg gcgaaggcgg ccccctggac ccccctggac aaagactgac aaagactgac 720 720 gctcaggtgc gaaagcgtgg gctcaggtgc gaaagcgtggggagcaaaca ggagcaaacaggattagata ggattagata ccctggtagt ccctggtagt ccacgccgta ccacgccgta 780 780 aacgatgtcg acttggaggt aacgatgtcg acttggaggttgtgcccttg tgtgcccttgaggcgtggct aggcgtggct tccggagcta tccggagcta acgcgttaag acgcgttaag 840 840 tcgaccgcct ggggagtacggccgcaaggt tcgaccgcct ggggagtacg gccgcaaggttaaaactcaa taaaactcaa atgaattgac atgaattgac gggggcccgc gggggcccgc 900 900 acaagcggtg gagcatgtgg acaagcggtg gagcatgtggtttaattcga tttaattcgatgcaacgcga tgcaacgcga agaaccttac agaaccttac ctactcttga ctactcttga 960 960 catccagaga acttagcaga catccagaga acttagcagagatgctttgg gatgctttggtgccttcggg tgccttcggg aactctgaga aactctgaga caggtgctgc caggtgctgc 1020 1020 atggctgtcg tcagctcgtg atggctgtcg tcagctcgtgttgtgaaatg ttgtgaaatgttgggttaag ttgggttaag tcccgcaacg tcccgcaacg agcgcaaccc agcgcaaccc 1080 1080 ttatcctttg ttgccagcgg tccggccggg ttatcctttg ttgccagcgg tccggccgggaactcaaagg aactcaaagg agactgccag agactgccag tgataaactg tgataaactg 1140 1140 gaggaaggtg gggatgacgt gaggaaggtg gggatgacgtcaagtcatca caagtcatcatggcccttac tggcccttac gagtagggct gagtagggct acacacgtgc acacacgtgc 1200 1200 tacaatggca tatacaaaga tacaatggca tatacaaagagaagcgacct gaagcgacctcgcgagagca cgcgagagca agcggacctc agcggacctc ataaagtatg ataaagtatg 1260 1260 tcgtagtccg gattggagtc tcgtagtccg gattggagtctgcaactcga tgcaactcgactccatgaag ctccatgaag tcggaatcgc tcggaatcgc tagtaatcgt tagtaatcgt 1320 1320 agatcagaat gctacggtga agatcagaat gctacggtgaatacgttccc atacgttcccgggccttgta gggccttgta cacaccgccc cacaccgccc gtcacaccat gtcacaccat 1380 1380 gggagtgggt tgcaaaagaa gggagtgggt tgcaaaagaa gtaggtagct gtaggtagct taaccttcgg taaccttcgg gaggncgctt gaggncgctt taccactt taccactt 1438 1438
<210> <210> 12 12 <211> <211> 1511 1511 <212> <212> DNA DNA <213> <213> Enterobacter cloacae Enterobacter cloacae
<400> 12 <400> 12 tgaacgctgg cggcaggcct tgaacgctgg cggcaggcctaacacatgca aacacatgcaagtcgaacgg agtcgaacgg tagcacagag tagcacagag agcttgctct agcttgctct
cgggtgacga gtggcggacg cgggtgacga gtggcggacgggtgagtaat ggtgagtaatgtctgggaaa gtctgggaaa ctgcctgatg ctgcctgatg gagggggata gagggggata 120 120
actactggaa acggtagcta actactggaa acggtagctaataccgcata ataccgcataaygtcgcaag aygtcgcaag accaaagagg accaaagagg gggaccttcg gggaccttcg 180 180
ggcctcttgc catcagatgt ggcctcttgc catcagatgtgcccagatgg gcccagatgggattagctag gattagctag taggtggggt taggtggggt aacggctcac aacggctcac 240 240
ctaggcgacg atccctagct ggtctgagag ctaggcgacg atccctagct ggtctgagaggatgaccage gatgaccagc cacactggaa cacactggaa ctgagacacg ctgagacacg 300 300
gtccagactc ctacgggagg gtccagactc ctacgggaggcagcagtggg cagcagtggggaatattgca gaatattgca caatgggcgc caatgggcgc aagcctgatg aagcctgatg 360 360
cagccatgcc gcgtgtatga cagccatgcc gcgtgtatgaagaaggcctt agaaggccttcgggttgtaa cgggttgtaa agtactttca agtactttca gcggggagga gcggggagga 420 420
aggtgttgtg gttaataacc aggtgttgtg gttaataaccgcagcaattg gcagcaattgacgttacccg acgttacccg cagaagaagc cagaagaage accggctaac accggctaac 480 480
tccgtgccag cagccgcggt tccgtgccag cagccgcggtaatacggagg aatacggagggtgcaagcgt gtgcaagcgt taatcggaat taatcggaat tactgggcgt tactgggcgt 540 540
aaagcgcacg caggcggtct aaagcgcacg caggcggtctgtcaagtcgg gtcaagtcggatgtgaaatc atgtgaaatc cccgggctca cccgggctca acctgggaac acctgggaac 600 600
tgcattcgaa actggcaggc tgcattcgaa actggcaggctggagtcttg tggagtcttgtagagggggg tagagggggg tagaattcca tagaattcca ggtgtagcgg ggtgtagcgg 660 660
tgaaatgcgt agagatctgg tgaaatgcgt agagatctggaggaataccg aggaataccggtggcgaagg gtggcgaagg cggccccctg cggccccctg gacaaagact gacaaagact 720 720
gacgctcagg tgcgaaagcg gacgctcagg tgcgaaagcgtggggagcaa tggggagcaaacaggattag acaggattag ataccctggt ataccctggt agtccacgcc agtccacgcc 780 780
gtaaacgatg tcgatttgga gtaaacgatg tcgatttggaggttgtgccc ggttgtgcccttgaggcgtg ttgaggcgtg gcttccggag gcttccggag ctaacgcgtt ctaacgcgtt 840 840
aaatcgaccg cctggggagt aaatcgaccg cctggggagtacggccgcaa acggccgcaaggttaaaact ggttaaaact caaatgaatt caaatgaatt gacgggggcc gacgggggcc 900 cgcacaagcg gtggagcatg cgcacaagcg gtggagcatgtggtttaatt tggtttaattcgatgcaacg cgatgcaacg cgaagaacct cgaagaacct tacctggtct tacctggtct 960 960 tgacatccac agaactttcc tgacatccac agaactttccagagatggat agagatggattggtgccttc tggtgccttc gggaactgtg gggaactgtg agacaggtgc agacaggtgc 1020 1020 tgcatggctg tcgtcagctc tgcatggctg tcgtcagctcgtgttgtgaa gtgttgtgaaatgttgggtt atgttgggtt aagtcccgca aagtcccgca acgagcgcaa acgagcgcaa 1080 1080 cccttatcct ttgttgccag cccttatcct ttgttgccagcggtccggcc cggtccggccgggaactcaa gggaactcaa aggagactgc aggagactgc cagtgataaa cagtgataaa 1140 1140 ctggaggaag gtggggatgacgtcaagtca ctggaggaag gtggggatga cgtcaagtcatcatggccct tcatggccct tacgaccagg tacgaccagg gctacacacg gctacacacg 1200 1200 tgctacaatg gcgcatacaa tgctacaatg gcgcatacaaagagaagcga agagaagcgacctcgcgaga cctcgcgaga gcaagcggac gcaagcggac ctcataaagt ctcataaagt 1260 1260 gcgtcgtagt ccggattgga gcgtcgtagt ccggattggagtctgcaact gtctgcaactcgactccatg cgactccatg aagtcggaat aagtcggaat cgctagtaat cgctagtaat 1320 1320 cgtagatcag aatgctacgg cgtagatcag aatgctacggtgaatacgtt tgaatacgttcccgggcctt cccgggcctt gtacacaccg gtacacaccg cccgtcacac cccgtcacac 1380 1380 catgggagtg ggttgcaaaa catgggagtg ggttgcaaaagaagtaggta gaagtaggtagcttaacctt gcttaacctt cgggagggcg cgggagggcg cttaccactt cttaccactt 1440 1440 tgtgattcat gactggggtg tgtgattcat gactggggtgaagtcgtaac aagtcgtaacaaggtaaccg aaggtaaccg taggggaacc taggggaacc tgcggctgga tgcggctgga 1500 1500 t c a c c t c c t t g g t C a C C t C C t t 1511 1511
<210> <210> 13 13 <211> <211> 1534 1534 <212> <212> DNA DNA <213> <213> Klebsiella pneumoniae Klebsiella pneumoniae
<220> <220> <221> <221> misc_feature misc_feature <222> <222> (11)..(12) (11)..(12) <223> <223> n is a, n is a, C, c,g,g,orort t
<400> 13 <400> 13 agagtttgat nntggctcag agagtttgat nntggctcagattgaacgct attgaacgctggcggcaggc ggcggcaggc ctaacacatg ctaacacatg caagtcgagc caagtcgage
ggtagcacag agagcttgct ggtagcacag agagcttgctctcgggtgac ctcgggtgacgagcggcgga gagcggcgga cgggtgagta cgggtgagta atgtctggga atgtctggga 120 120
aactgcctga tggaggggga aactgcctga tggagggggataactactgg taactactggaaacggtagc aaacggtagc taataccgca taataccgca taacgtcgca taacgtcgca 180 180
agaccaaagt gggggacctt agaccaaagt gggggaccttcgggcctcat cgggcctcatgccatcagat gccatcagat gtgcccagat gtgcccagat gggattagct gggattagct 240 240
agtaggtggg gtaacggctc agtaggtggg gtaacggctcacctaggcga acctaggcgacgatccctag cgatccctag ctggtctgag ctggtctgag aggatgacca aggatgacca 300 300
gccacactgg aactgagaca gccacactgg aactgagacacggtccagac cggtccagactcctacggga tcctacggga ggcagcagtg ggcagcagtg gggaatattg gggaatattg 360 360
cacaatgggc gcaagcctga cacaatgggc gcaagcctgatgcagccatg tgcagccatgccgcgtgtgt ccgcgtgtgt gaagaaggcc gaagaaggcc ttcgggttgt ttcgggttgt 420 420
aaagcacttt cagcggggag aaagcacttt cagcggggaggaaggcgatg gaaggcgatgaggttaataa aggttaataa cctcatcgat cctcatcgat tgacgttacc tgacgttacc 480 480
ctgcagaaga agcaccggct ctgcagaaga agcaccggctaactccgtgc aactccgtgccagcagccgc cagcagccgc ggtaatacgg ggtaatacgg agggtgcaag agggtgcaag 540 540
cgttaatcgg aattactggg cgttaatcgg aattactgggcgtaaagcgc cgtaaagcgcacgcaggcgg acgcaggcgg tctgtcaagt tctgtcaagt cggatgtgaa cggatgtgaa 600 600
atccccgggc tcaacctggg atccccgggc tcaacctgggaactgcattc aactgcattcgaaactggca gaaactggca ggctagagtc ggctagagtc ttgtagaggg ttgtagaggg 660 660
gggtagaatt ccaggtgtag gggtagaatt ccaggtgtagcggtgaaatg cggtgaaatgcgtagagatc cgtagagatc tggaggaata tggaggaata ccggtggcga ccggtggcga 720 720
aggcggcccc ctggacaaag aggcggcccc ctggacaaagactgacgctc actgacgctcaggtgcgaaa aggtgcgaaa gcgtggggag gcgtggggag caaacaggat caaacaggat 780 780
tagataccct ggtagtccac tagataccct ggtagtccacgccgtaaacg gccgtaaacgatgtcgattt atgtcgattt ggaggttgtg ggaggttgtg cccttgaggc cccttgaggc 840 840
gtggcttccg gagctaacgc gtggcttccg gagctaacgcgttaaatcga gttaaatcgaccgcctgggg ccgcctgggg agtacggccg agtacggccg caaggttaaa caaggttaaa 900 actcaaatga attgacgggg actcaaatga attgacgggggcccgcacaa gcccgcacaagcggtggagc gcggtggagc atgtggttta atgtggttta attcgatgca attcgatgca 960 960 acgcgaagaa ccttacctgg tcttgacate acgcgaagaa ccttacctgg tcttgacatccacagaactt cacagaactt tccagagatg tccagagatg gattggtgcc gattggtgcc 1020 1020 ttcgggaact gtgagacagg tgctgcatgg ttcgggaact gtgagacagg tgctgcatggctgtcgtcag ctgtcgtcag ctcgtgttgt ctcgtgttgt gaaatgttgg gaaatgttgg 1080 1080 gttaagtccc gcaacgagcg gttaagtccc gcaacgagcgcaacccttat caacccttatcctttgttgc cctttgttgc cagcggttag cagcggttag gccgggaact gccgggaact 1140 1140 caaaggagac tgccagtgat caaaggagac tgccagtgataaactggagg aaactggaggaaggtgggga aaggtgggga tgacgtcaag tgacgtcaag tcatcatggc tcatcatggc 1200 1200 ccttacgacc agggctacac ccttacgace agggctacacacgtgctaca acgtgctacaatggcatata atggcatata caaagagaag caaagagaag cgacctcgcg cgacctcgcg 1260 1260 agagcaagcg gacctcataa agagcaagcg gacctcataaagtatgtcgt agtatgtcgtagtccggatt agtccggatt ggagtctgca ggagtctgca actcgactcc actcgactcc 1320 1320 atgaagtcgg aatcgctagt atgaagtcgg aatcgctagtaatcgtagat aatcgtagatcagaatgcta cagaatgcta cggtgaatac cggtgaatac gttcccgggc gttcccgggc 1380 1380 cttgtacaca ccgcccgtca cttgtacaca ccgcccgtcacaccatggga caccatgggagtgggttgca gtgggttgca aaagaagtag aaagaagtag gtagcttaac gtagcttaac 1440 1440 cttcgggagg gcgcttacca cttcgggagg gcgcttaccactttgtgatt ctttgtgattcatgactggg catgactggg gtgaagtcgt gtgaagtcgt aacaaggtaa aacaaggtaa 1500 1500 ccgtagggga acctgcggtt ggatcacctc cttt ccgtagggga 1534 acctgcggtt ggatcaccto cttt 1534
<210> <210> 14 14 <211> <211> 1536 1536 <212> <212> DNA DNA <213> <213> Pseudomonasaeruginosa Pseudomonas aeruginosa
<400> <400> 14 14 gaactgaaga gtttgatcatggctcagatt gaactgaaga gtttgatcat ggctcagattgaacgctggc gaacgctggc ggcaggccta ggcaggccta acacatgcaa acacatgcaa
gtcgagcgga tgaagggage gtcgagcgga tgaagggagcttgctcctgg ttgctcctggattcagcggc attcagcggc ggacgggtga ggacgggtga gtaatgccta gtaatgccta 120 ggaatctgcc tggtagtggg ggaatctgcc tggtagtgggggataacgtc ggataacgtccggaaacggg cggaaacggg cgctaatacc cgctaatacc gcatacgtcc gcatacgtcc 180 180 tgagggagaa agtgggggat tgagggagaa agtgggggatcttcggacct cttcggacctcacgctatca cacgctatca gatgagccta gatgagecta ggtcggatta ggtcggatta 240 240 gctagttggt ggggtaaagg gctagttggt ggggtaaaggcctaccaagg cctaccaaggcgacgatccg cgacgatccg taactggtct taactggtct gagaggatga gagaggatga 300 300 tcagtcacac tggaactgag tcagtcacac tggaactgagacacggtcca acacggtccagactcctacg gactcctacg ggaggcagca ggaggcagca gtggggaata gtggggaata 360 360 ttggacaatg ggcgaaagcc ttggacaatg ggcgaaagcctgatccagcc tgatccagccatgccgcgtg atgccgcgtg tgtgaagaag tgtgaagaag gtcttcggat gtcttcggat 420 420 tgtaaagcac tttaagttgg tgtaaagcac tttaagttgggaggaagggc gaggaagggcagtaagttaa agtaagttaa taccttgctg taccttgctg ttttgacgtt ttttgacgtt 480 480 accaacagaa taagcaccgg accaacagaa taagcaccggctaacttcgt ctaacttcgtgccagcagcc gccagcagcc gcggtaatac gcggtaatac gaagggtgca gaagggtgca 540 540 agcgttaatc ggaattactg agcgttaatc ggaattactgggcgtaaagc ggcgtaaagcgcgcgtaggt gcgcgtaggt ggttcagcaa ggttcagcaa gttggatgtg gttggatgtg 600 600 aaatccccgg gctcaacctg aaatccccgg gctcaacctgggaactgcat ggaactgcatccaaaactac ccaaaactac tgagctagag tgagctagag tacggtagag tacggtagag 660 660 ggtggtggaa tttcctgtgt ggtggtggaa tttcctgtgtagcggtgaaa agcggtgaaatgcgtagata tgcgtagata taggaaggaa taggaaggaa caccagtggc caccagtggc 720 720 gaaggcgacc acctggactg gaaggcgace acctggactgatactgacac atactgacactgaggtgcga tgaggtgcga aagcgtgggg aagcgtgggg agcaaacagg agcaaacagg 780 780 attagatacc ctggtagtcc attagatacc ctggtagtccacgccgtaaa acgccgtaaacgatgtcgac cgatgtcgac tagccgttgg tagccgttgg gatccttgag gatccttgag 840 840 atcttagtgg cgcagctaac atcttagtgg cgcagctaacgcgataagtc gcgataagtcgaccgcctgg gaccgcctgg ggagtacggc ggagtacggc cgcaaggtta cgcaaggtta 900 900 aaactcaaat gaattgacgg aaactcaaat gaattgacgggggcccgcac gggcccgcacaagcggtgga aagcggtgga gcatgtggtt gcatgtggtt taattcgaag taattcgaag 960 960 caacgcgaag aaccttacctggccttgaca caacgcgaag aaccttacct ggccttgacatgctgagaac tgctgagaac tttccagaga tttccagaga tggattggtg tggattggtg 1020 ccttcgggaa ctcagacaca ccttcgggaa ctcagacacaggtgctgcat ggtgctgcatggctgtcgtc ggctgtcgtc agctcgtgtc agctcgtgtc gtgagatgtt gtgagatgtt 1080 1080 gggttaagtc ccgtaacgag gggttaagtc ccgtaacgagcgcaaccctt cgcaacccttgtccttagtt gtccttagtt accagcacct accagcacct cgggtgggca cgggtgggca 1140 1140 ctctaaggag actgccggtg acaaaccgga ctctaaggag actgccggtg acaaaccggaggaaggtggg ggaaggtggg gatgacgtca gatgacgtca agtcatcatg agtcatcatg 1200 1200 gcccttacgg ccagggctac gcccttacgg ccagggctacacacgtgcta acacgtgctacaatggtcgg caatggtcgg tacaaagggt tacaaaaggt tgccaagccg tgccaagccg 1260 1260 cgaggtggag ctaatcccat cgaggtggag ctaatcccataaaaccgatc aaaaccgatcgtagtccgga gtagtccgga tcgcagtctg tcgcagtctg caactcgact caactcgact 1320 1320 gcgtgaagtc ggaatcgcta gcgtgaagtc ggaatcgctagtaatcgtga gtaatcgtgaatcagaatgt atcagaatgt cacggtgaat cacggtgaat acgttcccgg acgttcccgg 1380 1380 gccttgtaca caccgcccgt gccttgtaca caccgcccgtcacaccatgg cacaccatgggagtgggttg gagtgggttg ctccagaagt ctccagaagt agctagtcta agctagtcta 1440 1440 accgcaaggg ggacggttac accgcaaggg ggacggttaccacggagtga cacggagtgattcatgactg ttcatgactg gggtgaagtc gggtgaagtc gtaacaaggt gtaacaaggt 1500 1500 agccgtaggg gaacctgcgg ctggatcacc tcctta agccgtaggg 1536 gaacctgcgg ctggatcacc tcctta 1536
<210> <210> 15 15 <211> <211> 1497 1497 <212> <212> DNA DNA <213> <213> Acinetobacter calcoaceticus Acinetobacter calcoaceticus
<400> <400> 15 15 tagagtttga tcctggctcagattgaacgc tagagtttga tcctggctca gattgaacgctggcggcagg tggcggcagg cttaacacat cttaacacat gcaagtcgag gcaagtcgag
cggggaaagg tagcttgcta cggggaaagg tagcttgctactggacctag ctggacctagcggcggacgg cggcggacgg gtgagtaatg gtgagtaatg cttaggaatc cttaggaato 120 120
tgcctattag tgggggacaa tgcctattag tgggggacaacattccgaaa cattccgaaaggaatgctaa ggaatgctaa taccgcatac taccgcatac gtcctacggg gtcctacggg 180 180
agaaagcagg ggaccttcgg agaaagcagg ggaccttcgggccttgcgct gccttgcgctaatagatgag aatagatgag cctaagtcgg cctaagtcgg attagctagt attagctagt 240 tggtggggta aaggcctacc tggtggggta aaggcctaccaaggcgacga aaggcgacgatctgtagcgg tctgtagcgg tctgagagga tctgagagga tgatccgcca tgatccgcca 300 300 cactgggact gagacacggc cactgggact gagacacggcccagactect ccagactcctacgggaggca acgggaggca gcagtgggga gcagtgggga atattggaca atattggaca 360 360 atggggggaa ccctgatcca atggggggaa ccctgatccagccatgccgc gccatgccgcgtgtgtgaag gtgtgtgaag aaggccttat aaggccttat ggttgtaaag ggttgtaaag 420 420 cactttaagc gaggaggagg cactttaage gaggaggaggctactagtat ctactagtattaatactact taatactact ggatagtgga ggatagtgga cgttactcgc cgttactcgc 480 480 agaataagca ccggctaact agaataagca ccggctaactctgtgccagc ctgtgccagcagccgcggta agccgcggta atacagaggg atacagaggg tgcgagcgtt tgcgagcgtt 540 540 aatcggattt actgggcgtaaagcgtgcgt aatcggattt actgggcgta aagcgtgcgtaggcggccat aggcggccat ttaagtcaaa ttaagtcaaa tgtgaaatcc tgtgaaatcc 600 600 ccgagcttaa cttgggaatt ccgagcttaa cttgggaattgcattgcata gcattgcatactggatggct ctggatggct agagtatggg agagtatggg agaggatggt agaggatggt 660 660 agaattccag gtgtagcggt agaattccag gtgtagcggtgaaatgcgta gaaatgcgtagagatctgga gagatctgga ggaataccga ggaataccga tggcgaaggc tggcgaaggc 720 720 agccatctgg cctaatactg agccatctgg cctaatactgacgctgaggt acgctgaggtacgaaagcat acgaaagcat gggagcagaa gggagcagaa caggattaga caggattaga 780 780 taccctggta gtccatgccg taccctggta gtccatgccgtaaacgatgt taaacgatgtttactagccg ttactagccg ttggggcctt ttggggcctt tgaggcttta tgaggcttta 840 840 gtggcgcagc taacgcgata gtggcgcagc taacgcgataagtagaccgc agtagaccgcctggggagta ctggggagta cggtcgcaag cggtcgcaag actaaaactc actaaaactc 900 900 aaatgaattg acgggggccc aaatgaattg acgggggcccgcacaagcgg gcacaagcggtggagcatgt tggagcatgt ggtttaattc ggtttaattc gatgcaacgc gatgcaacgc 960 960 gaagaacctt acctggcctt gaagaacctt acctggccttgacatactag gacatactagaaactttcca aaactttcca gagatggatt gagatggatt ggtgccttcg ggtgccttcg 1020 1020 ggaatttaga tacaggtgct ggaatttaga tacaggtgctgcatggctgt gcatggctgtcgtcagctcg cgtcagctcg tgtcgtgaga tgtcgtgaga tgttgggtta tgttgggtta 1080 1080 agtcccgcaa cgagcgcaac agtcccgcaa cgagcgcaacccttttcctt ccttttccttacttgccagc acttgccagc atttcggatg atttcggatg ggaactttaa ggaactttaa 1140 ggatactgcc agtgacaaac ggatactgcc agtgacaaactggaggaagg tggaggaaggcggggacgac cggggacgac gtcaagtcat gtcaagtcat catggccctt catggccctt 1200 1200 acggccaggg ctacacacgt acggccaggg ctacacacgtgctacaatgg gctacaatggtcggtacaaa tcggtacaaa gggttgctac gggttgctac ctagcgatag ctagcgatag 1260 1260 gatgctaatc tcaaaaagcc gatgctaatc tcaaaaagccgatcgtagto gatcgtagtccggattggag cggattggag tctgcaactc tctgcaactc gactccatga gactccatga 1320 1320 agtcggaatc gctagtaatc agtcggaatc gctagtaatcgcggatcaga gcggatcagaatgcccggtg atgcccggtg atacgttccc atacgttccc gggccttgta gggccttgta 1380 1380 cacaccgccc gtcacaccat gggagtttgt cacaccgccc gtcacaccat gggagtttgttgcaccagaa tgcaccagaa gtaggtagtc gtaggtagtc taaccgcaag taaccgcaag 1440 1440 gaggacgctt accacggtgt gaggacgctt accacggtgt ggccgatgac ggccgatgac tggggtgaag tggggtgaag tcgtaacaag tcgtaacaaggtaacca gtaacca 1497 1497
<210> <210> 16 16 <211> <211> 19 19 <212> <212> DNA DNA <213> <213> ArtificialSequence Artificial Sequence
<220> <220> <223> Forward Primer <223> Forward Primer
<400> 16 <400> 16 c c t c t t g c c a t c g g a t g t g cctcttgcca 19 19 tcggatgtg <210> <210> 17 17 <211> <211> 20 20 <212> <212> DNA DNA <213> <213> ArtificialSequence Artificial Sequence
<220> <220> <223> Reverse Primer <223> Reverse Primer
<400> <400> 17 17 c c a g t g t g g c t g g t c a t c c t cagtgtggo
tggtcatcct
<210> <210> 18 18 <211> <211> 20 20 <212> <212> DNA DNA <213> <213> ArtificialSequence Artificial Sequence
<220> <220> <223> Forward Primer <223> Forward Primer
<400> <400> 18 18 c c t a c g g g a g g c a g c a g t a g cctacgggag
gcagcagtag <210> <210> 19 19 <211> <211> 20 20 <212> <212> DNA DNA <213> <213> Artificial Sequence Artificial Sequence
<220> <220> <223> ForwardPrimer <223> Forward Primer
<400> <400> 19 19 g g g a g g c a g c a g t a g g g a a t gggaggcagc
agtagggaat <210> <210> 20 20 <211> <211> 22 22 <212> <212> DNA DNA <213> <213> Artificial Sequence Artificial Sequence
<220> <220> <223> ReversePrimer <223> Reverse Primer
<400> <400> 20 20 c g a t c c g a a a a c c t t c t t c a c t cgatccgaaa 22 22 accttcttca ct
<210> <210> 21 21 <211> <211> 26 26 <212> <212> DNA DNA <213> <213> Artificial Sequence Artificial Sequence
<220> <220> <223> <223> Forward Primer Forward Primer
<400> <400> 21 21 a a g a c g g t c t t g c t g t c a c t t a t a g a aagacggtct 26 26 tgctgtcact tataga
<210> <210> 22 22 <211> <211> 22 22 <212> <212> DNA DNA <213> <213> ArtificialSequence Artificial Sequence
<220> <220> <223> ReversePrimer <223> Reverse Primer
<400> <400> 22 22 c t a t g c a t c g t t g c c t t g g t a a a a ctatgcatcg 22 22 ttgccttggt <210> <210> 23 23 <211> <211> 18 18 <212> <212> DNA DNA <213> <213> Artificial Sequence Artificial Sequence
<220> <220> <223> ForwardPrimer <223> Forward Primer
<400> <400> 23 23 t g c c g c g t g a a t g a a g a a tgccgcgtga 18 18 atgaagaa <210> <210> 24 24 <211> <211> 21 21 <212> <212> DNA DNA <213> <213> ArtificialSequence Artificial Sequence
<220> <220> <223> ForwardPrimer <223> Forward Primer
<400> <400> 24 24 g c g t g a a g g a t g a a g g c t c t a a gcgtgaagga 21 21 tgaaggctct
<210> <210> 25 25 <211> <211> 21 21 <212> <212> DNA DNA <213> <213> Artificial Sequence Artificial Sequence
<220> <220> <223> ForwardPrimer <223> Forward Primer
<400> <400> 25 25 t g a t g a a g g t t t t c g g a t c g t t tgatgaaggt 21 21 ttcggatcg <210> <210> 26 26 <211> <211> 29 29 <212> <212> DNA DNA <213> <213> ArtificialSequence Artificial Sequence
<220> <220> <223> ReversePrimer <223> Reverse Primer
<400> <400> 26 26 tgatgtacta ttaacacatc aaccttcct tgatgtacta 29 29 Etaacacat aaccttcct
<210> <210> 27 27 <211> <211> 22 22 <212> <212> DNA DNA <213> <213> Artificial Sequence Artificial Sequence
<220> <220> <223> Reverse Primer <223> Reverse Primer
<400> <400> 27 27 a a c g c t c g g a t c t t c c g t a t t a t a aacgctcga 22 22 tcttccgtat <210> <210> 28 28 <211> <211> 21 21 <212> DNA <212> DNA <213> ArtificialSequence <213> Artificial Sequence
<220> <220> <223> <223> Reverse Primer Reverse Primer
<400> <400> 28 28 c g c t c g c c a c c t a c g t a t t a c C cgctcgccac 21 21 ctacgtatta <210> <210> 29 29 <211> <211> 28 28 <212> <212> DNA DNA <213> <213> Artificial Sequence Artificial Sequence
<220> <220> <223> ForwardPrimer <223> Forward Primer
<400> <400> 29 29 g t t g t a a g a g a a g a a c g a g t g t g a g a g t gttgtaagag 28 28 aagaacgagt gtgagagt
<210> <210> 30 30 <211> <211> 22 22 <212> <212> DNA DNA <213> <213> ArtificialSequence Artificial Sequence
<220> <220> <223> Reverse Primer <223> Reverse Primer
<400> <400> 30 30 c g t a g t t a g c c g t c c c t t t c t g cgtagttaga 22 22 cgtccctttc tg
<210> <210> 31 31 <211> <211> 24 24 <212> <212> DNA DNA <213> <213> Artificial Sequence Artificial Sequence
<220> <220> <223> Forward Primer <223> Forward Primer
<400> <400> 31 31 g c g g t t t g t t a a g t c a g a t g t t g g a a a a gcggtttgtt 24 24 aagtcagatg
<210> <210> 32 32 <211> <211> 22 22 <212> <212> DNA DNA <213> <213> Artificial Sequence Artificial Sequence
<220> <220> <223> ForwardPrimer <223> Forward Primer
<400> <400> 32 32 g g t c t g t c a a g t c g g a t g t g a a a a ggtctgtcaa 22 22 gtcggatgtg <210> <210> 33 33 <211> <211> 21 21 <212> <212> DNA DNA <213> <213> ArtificialSequence Artificial Sequence
<220> <220> <223> Forward Primer <223> Forward Primer
<400> <400> 33 33 t c a a c c t g g g a a c t c a t t c g a a tcaacctggg 21 21 aactcattcg <210> <210> 34 34 <211> <211> 22 22 <212> <212> DNA DNA <213> <213> ArtificialSequence Artificial Sequence
<220> <220> <223> Reverse Primer <223> Reverse Primer
<400> <400> 34 34 g g a a t t c t a c c c c c c t c t a c g a g a ggaattctac 22 22 Ccccctcta C
<210> <210> 35 35 <211> <211> 23 23 <212> <212> DNA DNA <213> <213> ArtificialSequence Artificial Sequence
<220> <220> <223> <223> Reverse Primer Reverse Primer
<400> <400> 35 35 g g a a t t c t a c c c c c c t c t a c a a gg a a ggaattctac 23 23 Ccccctctaa <210> <210> 36 36 <211> <211> 1720 1720 <212> <212> DNA DNA <213> <213> Aspergillusfumigatus Aspergillus fumigatus
<400> <400> 36 36 ggaccgggcc tgtctaggtataagcaattt ggaccgggcc tgtctaggta taagcaatttatacggtgaa atacggtgaa actgcgaatg actgcgaatg gctcattaaa gctcattaaa
tcagttatcg tttatttgat agtaccttac tcagttatcg tttatttgat agtaccttactacatggata tacatggata cctgtggtaa cctgtggtaa ttctagagct ttctagagct 120 120
aatacatgct aaaaacctcg aatacatgct aaaaacctcgacttcggaag acttcggaaggggtgtattt gggtgtattt attagataaa attagataaa aaaccaatgc aaaccaatgc 180 180
ccttcggggc tccttggtgaatcataataa ccttcggggc tccttggtga atcataataacttaacgaat cttaacgaat cgcatggcct cgcatggcct tgcgccggcg tgcgccggcg 240 240
atggttcatt caaatttctg atggttcatt caaatttctgccctatcaac ccctatcaactttcgatggt tttcgatggt aggatagtgg aggatagtgg cctaccatgg cctaccatgg 300 300
tggcaacggg taacggggaa ttagggttcg tggcaaccgg taacggggaa ttagggttcgattccggaga attccggaga gggagcctga gggagcctga gaaacggcta gaaacggcta 360 360
ccacatccaa ggaaggcage ccacatccaa ggaaggcagcaggcgcgcaa aggcgcgcaaattacccaat attacccaat cccgacacgg cccgacacgg ggaggtagtg ggaggtagtg 420 420
acaataaata ctgatacggg acaataaata ctgatacggggctcttttgg gctcttttgggtctcgtaat gtctcgtaat tggaatgagt tggaatgagt acaatctaaa acaatctaaa 480 480
tcccttaacg aggaacaatt tcccttaacg aggaacaattggagggcaag ggagggcaagtctggtgcca tctggtgcca gcagccgcgg gcagccgcgg taattccagc taattccagc 540 540
tccaatagcg tatattaaag ttgttgcagt tccaatagcg tatattaaag ttgttgcagttaaaaagctc taaaaagctc gtagttgaac gtagttgaac cttgggtctg cttgggtctg 600 gctggccggt ccgcctcacc gctggccggt ccgcctcaccgcgagtactg gcgagtactggtccggctgg gtccggctgg acctttcctt acctttcctt ctggggaacc ctggggaacc 660 660 tcatggcctt cactggctgt tcatggcctt cactggctgtggggggaacc ggggggaaccaggactttta aggactttta ctgtgaaaaa ctgtgaaaaa attagagtgt attagagtgt 720 720 tcaaagcagg cctttgctcg aatacattag tcaaagcagg cctttgctcg aatacattagcatggaataa catggaataa tagaatagga tagaatagga cgtgcggttc cgtgcggttc 780 780 tattttgttg gtttctagga ccgccgtaat tattttgttg gtttctagga ccgccgtaatgattaatagg gattaatagg gatagtcggg gatagtcggg ggcgtcagta ggcgtcagta 840 840 ttcagctgtc agaggtgaaattcttggatt ttcagctgtc agaggtgaaa ttcttggatttgctgaagac tgctgaagac taactactgc taactactgc gaaagcattc gaaagcattc 900 900 gccaaggatg ttttcattaa gccaaggatg ttttcattaatcaggaacga tcaggaacgaaagttagggg aagttagggg atcgaagacg atcgaagacg atcagatacc atcagatacc 960 960 gtcgtagtct taaccataaa gtcgtagtct taaccataaactatgccgac ctatgccgactagggatcgg tagggatcgg gcggtgtttc gcggtgtttc tatgatgacc tatgatgace 1020 1020 cgctcggcac cttacgagaa cgctcggcac cttacgagaaatcaaagttt atcaaagtttttgggttctg ttgggttctg gggggagtat gggggagtat ggtcgcaagg ggtcgcaagg 1080 1080 ctgaaactta aagaaattga ctgaaactta aagaaattgacggaagggca cggaagggcaccacaaggcg ccacaaggcg tggagcctgc tggagcctgc ggcttaattt ggcttaattt 1140 1140 gactcaacac ggggaaactc gactcaacac ggggaaactcaccaggtcca accaggtccagacaaaataa gacaaaataa ggattgacag ggattgacag attgagagct attgagagct 1200 1200 ctttcttgat cttttggatg ctttcttgat cttttggatggtggtgcatg gtggtgcatggccgttctta gccgttctta gttggtggag gttggtggag tgatttgtct tgatttgtct 1260 1260 gcttaattgc gataacgaac gcttaattgc gataacgaacgagacctcgg gagacctcggcccttaaata cccttaaata gcccggtccg gcccggtccg catttgcggg catttgcggg 1320 1320 ccgctggctt cttaggggga ccgctggctt cttagggggactatcggctc ctatcggctcaagccgatgg aagccgatgg aagtgcgcgg aagtgcgcgg caataacagg caataacagg 1380 1380 tctgtgatgc ccttagatgt tctgtgatgc ccttagatgttctgggccgc tctgggccgcacgcgcgcta acgcgcgcta cactgacagg cactgacagg gccagcgagt gccagcgagt 1440 1440 acatcacctt ggccgagagg acatcacctt ggccgagaggtctgggtaat tctgggtaatcttgttaaac cttgttaaac cctgtcgtgc cctgtcgtgc tggggataga tggggataga 1500 gcattgcaat tattgctctt gcattgcaat tattgctcttcaacgaggaa caacgaggaatgcctagtag tgcctagtag gcacgagtca gcacgagtca tcagctcgtg tcagctcgtg 1560 1560 ccgattacgt ccctgccctt ccgattacgt ccctgccctttgtacacacc tgtacacaccgcccgtcgct gcccgtcgct actaccgatt actaccgatt gaatggctcg gaatggctcg 1620 1620 gtgaggcctt cggactggct gtgaggcctt cggactggctcaggggagtt caggggagttggcaacgact ggcaacgact ccccagagcc ccccagagcc ggaaagttgg ggaaagttgg 1680 1680 tcaaacccgg tcattagagg aaagaaaaaa ttaaacacgg tcaaacccgg tcattagagg aaagaaaaaa ttaaacacgg 1720 1720
<210> <210> 37 37 <211> <211> 1632 1632 <212> <212> DNA DNA <213> <213> Candida albicans Candida albicans
<220> <220> <221> misc_feature <221> misc_feature <222> (1587)..(1587) <222> (1587)..(1587) <223> <223> nnis isa, a,C, c,g, g,orortt
<400> <400> 37 37 tcagttatcg tttatttgatagtaccttac tcagttatcg tttatttgat agtaccttactacttggata tacttggata accgtggtaa accgtggtaa ttctagagct ttctagagct
aatacatgct taaaatcccg aatacatgct taaaatcccgactgtttgga actgtttggaagggatgtat agggatgtat ttattagata ttattagata aaaaatcaat aaaaatcaat 120 120
gccttcgggc tctttgatga gccttcgggc tctttgatgattcataataa ttcataataacttttcgaat cttttcgaat cgcatggcct cgcatggcct tgtgctggcg tgtgctggcg 180 180
atggttcatt caaatttctg atggttcatt caaatttctgccctatcaac ccctatcaactttcgatggt tttcgatggt aggatagtgg aggatagtgg cctaccatgg cctaccatgg 240 240
tttcaacggg tttcaaccggg taacggggaa taagggttcg attccggaga taacggggaa taagggttcg attccggagagggagcctga gggagcctga gaaacggcta gaaacggcta 300 300
ccacatccaa ggaaggcage ccacatccaa ggaaggcagcaggcgcgcaa aggcgcgcaaattacccaat attacccaat cccgacacgg cccgacacgg ggaggtagtg ggaggtagtg 360 360
acaataaata acgatacagg acaataaata acgatacagggcccttttgg gcccttttgggtcttgtaat gtcttgtaat tggaatgagt tggaatgagt acaatgtaaa acaatgtaaa 420 taccttaacg aggaacaatt taccttaacg aggaacaattggagggcaag ggagggcaagtctggtgcca tctggtgcca gcagccgcgg gcagccgcgg taattccagc taattccagc 480 480 tccaaaagcg tatattaaagttgttgcagt tccaaaagcg tatattaaag ttgttgcagttaaaaagctc taaaaagctc gtagttgaac gtagttgaac cttgggcttg cttgggcttg 540 540 gctggccggt ccatcttttt gctggccggt ccatctttttgatgcgtact gatgcgtactggacccagcc ggacccagcc gagcctttcc gagcctttcc ttctgggtag ttctgggtag 600 600 ccatttatgg cgaaccagga ccatttatgg cgaaccaggacttttacttt cttttactttgaaaaaatta gaaaaaatta gagtgttcaa gagtgttcaa agcaggcctt agcaggcctt 660 660 tgctcgaata tattagcatg tgctcgaata tattagcatggaataataga gaataatagaataggacgtt ataggacgtt atggttctat atggttctat tttgttggtt tttgttggtt 720 720 tctaggacca tcgtaatgat taatagggac tctaggacca tcgtaatgat taatagggacggtcgggggt ggtcgggggt atcagtattc atcagtattc agttgtcaga agttgtcaga 780 780 ggtgaaattc ttggatttac ggtgaaattc ttggatttactgaagactaa tgaagactaactactgcgaa ctactgcgaa agcatttacc agcatttacc aaggacgttt aaggacgttt 840 840 tcattaatca agaacgaaagttaggggatc tcattaatca agaacgaaag ttaggggatcgaagatgatc gaagatgatc agataccgtc agataccgtc gtagtcttaa gtagtcttaa 900 900 ccataaacta tgccgactag ccataaacta tgccgactagggatcggttg ggatcggttgttgttctttt ttgttctttt attgacgcaa attgacgcaa tcggcacctt tcggcacctt 960 960 acgagaaatc aaagtctttg acgagaaatc aaagtctttgggttctgggg ggttctggggggagtatggt ggagtatggt cgcaaggctg cgcaaaggctg aaacttaaag aaacttaaag 1020 1020 gaattgacgg aagggcacca gaattgacgg aagggcaccaccaggagtgg ccaggagtggagcctgcggc agcctgcggc ttaatttgac ttaatttgac tcaacacggg tcaacacggg 1080 1080 gaaactcacc aggtccagac gaaactcacc aggtccagacacaataagga acaataaggattgacagatt ttgacagatt gagagctctt gagagctctt tcttgatttt tcttgatttt 1140 1140 gtgggtggtg gtgcatggcc gtgggtggtg gtgcatggccgttcttagtt gttcttagttggtggagtga ggtggagtga tttgtctgct tttgtctgct taattgcgat taattgcgat 1200 1200 aacgaacgag accttaacct aacgaacgag accttaacctactaaatagt actaaatagtgctgctagca gctgctagca tttgctggta tttgctggta tagtcacttc tagtcacttc 1260 1260 ttagagggac tatcgacttcaagtcgatgg ttagagggac tatcgacttc aagtcgatggaagtttgagg aagtttgagg caataacagg caataacagg tctgtgatgc tctgtgatgc 1320 ccttagacgt tctgggccgc ccttagacgt tctgggccgcacgcgcgcta acgcgcgctacactgacgga cactgacgga gccagcgagt gccagcgagt ataagccttg ataagccttg 1380 1380 gccgagaggt ctgggaaatc gccgagaggt ctgggaaatcttgtgaaact ttgtgaaactccgtcgtgct ccgtcgtgct ggggatagag ggggatagag cattgtaatt cattgtaatt 1440 1440 gttgctcttc aacgaggaat gttgctcttc aacgaggaattcctagtaag tcctagtaagcgcaaattcat cgcaagtcatcagcttgcgt cagcttgcgt tgattacgtc tgattacgtc 1500 1500 cctgcccttt gtacacaccg cctgcccttt gtacacaccgcccgtcgcta cccgtcgctactaccgattg ctaccgattg aatggcttag aatggcttag tgaggcctcc tgaggcctcc 1560 1560 ggattggttt aggaaagggg ggattggttt aggaaagggggcaactncat gcaactncattctggaaccg tctggaaccg agaagctggt agaagctggt caaacttggt caaacttggt 1620 1620 c a t t t a g a g g a a C a t t t a g a g g 1632 1632 a a
<210> <210> 38 38 <211> <211> 1732 1732 <212> <212> DNA DNA <213> <213> Candida glabrata Candida glabrata
<400> <400> 38 38 agtatttgtc taaaaattaagccatgcatg agtatttgtc taaaaattaa gccatgcatgtctaagtata tctaagtata agcaatttat agcaatttat acagtgaaac acagtgaaac
tgcgaatggc tcattaaatc tgcgaatggc tcattaaatcagttatcgtt agttatcgtttatttgatag tatttgatag ttcctttact ttcctttact acatggtata acatggtata 120 120
actgtggtaa ttctagagct actgtggtaa ttctagagctaatacatgct aatacatgcttaaaatctcg taaaatctcg acctcttgga acctcttgga agagatgtat agagatgtat 180 180
ttattagata aaaaatcaat gtcttcggac ttattagata aaaaatcaat gtcttcggactttttgatga tttttgatga ttcataataa ttcataataa cttttcgaat cttttcgaat 240 240
cgcatggcct tgtgctggcgatggttcatt cgcatggcct tgtgctggcg atggttcattcaaatttctg caaatttctg ccctatcaac ccctatcaac tttcgatggt tttcgatggt 300 300
aggatagtgg cctaccatgg aggatagtgg cctaccatggtttcaaccggg tttcaacggg taacggggaa taacggggaataagggttcg taagggttcg attccggaga attccggaga 360 360
gggagcctga gaaacggcta gggagcctga gaaacggctaccacatccaa ccacatccaaggaaggcage ggaaggcagc aggcgcgcaa aggcgcgcaa attacccaat attacccaat 420 cctgacacag ggaggtagtg cctgacacag ggaggtagtgacaataaata acaataaataacgatacagg acgatacagg gcccattcgg gcccattcgg gtcttgtaat gtcttgtaat 480 480 tggaatgagt acaatgtaaa tggaatgagt acaatgtaaataccttaacg taccttaacgaggaacaatt aggaacaatt ggagggcaag ggagggcaag tctggtgcca tctggtgcca 540 540 gcagccgcgg taattccago gcagccgcgg taattccagctccaatagcg tccaatagcgtatattaaag tatattaaag ttgttgcagt ttgttgcagt taaaaagctc taaaaagctc 600 600 gtagttgaac tttgggcctg gtagttgaac tttgggcctgggtggccggt ggtggccggtccgatttttt ccgatttttt cgtgtactgg cgtgtactgg aatgcacccg aatgcacccg 660 660 ggcctttcct tctggctaac ggcctttcct tctggctaaccccaaggtcct cccaagtcct tgtggcttgg tgtggcttggcggcgaacca cggcgaacca ggacttttac ggacttttac 720 720 tttgaaaaaa ttagagtgtt caaagcaggc tttgaaaaaa ttagagtgtt caaagcaggcgtattgctcg gtattgctcg aatatattag aatatattag catggaataa catggaataa 780 780 tggaatagga cgtttggttc tggaatagga cgtttggttctattttgttg tattttgttggtttctagga gtttctagga ccatcgtaat ccatcgtaat gattaatagg gattaatagg 840 840 gacggtcggg ggcatcagta gacggtcggg ggcatcagtattcaattgtc ttcaattgtcagaggtgaaa agaggtgaaa ttcttggatt ttcttggatt tattgaagac tattgaagac 900 900 taactactgc gaaagcattt taactactgc gaaagcatttgccaaaggacg gccaaggacg ttttcattaa ttttcattaatcaagaacga tcaagaacga aagttagggg aagttagggg 960 960 atcgaagatg atcagataccgtcgtagtct atcgaagatg atcagatacc gtcgtagtcttaaccataaa taaccataaa ctatgccgac ctatgccgac tagggatcgg tagggatcgg 1020 1020 gtggtgtttt tttagtgace gtggtgtttt tttagtgacccactcggcac cactcggcaccttacgagaa cttacgagaa atcaaagtct atcaaagtct ttgggttctg ttgggttctg 1080 1080 gggggagtat ggtcgcaagg gggggagtat ggtcgcaaggctgaaactta ctgaaacttaaaggaattga aaggaattga cggaagggca cggaagggca ccaccaggag ccaccaggag 1140 1140 tggagcctgc ggcttaattt tggagcctgc ggcttaatttgactcaacac gactcaacacggggaaactc ggggaaactc accaggtcca accaggtcca gacacaataa gacacaataa 1200 1200 ggattgacag attgagagct ggattgacag attgagagctctttcttgat ctttcttgattttgtgggtg tttgtgggtg gtggtgcatg gtggtgcatg gccgttctta gccgttctta 1260 1260 gttggtggag tgatttgtct gttggtggag tgatttgtctgcttaattgc gcttaattgcgataacgaac gataacgaac gagaccttaa gagaccttaa cctactaaat cctactaaat 1320 agtggtgcta gcatttgctg agtggtgcta gcatttgctggttgtccact gttgtccacttcttagaggg tcttagaggg actatcggtt actatcggtt tcaagccgat tcaagccgat 1380 1380 ggaagtttga ggcaataaca ggaagtttga ggcaataacaggtctgtgat ggtctgtgatgcccttagac gcccttagac gttctgggcc gttctgggcc gcacgcgcgc gcacgcgcgc 1440 1440 tacactgacg gagccagcga gtctaacctt tacactgacg gagccagcga gtctaaccttggccgagagg ggccgagagg tcttggtaat tcttggtaat cttgtgaaac cttgtgaaac 1500 1500 tccgtcgtgc tggggataga tccgtcgtgc tggggatagagcattgtaat gcattgtaattattgctctt tattgctctt caacgaggaa caacgaggaa ttcctagtaa ttcctagtaa 1560 1560 gcgcaagtca tcagcttgcg gcgcaaattca ttgattacgt ccctgccctt tcagcttgcg ttgattacgt ccctgccctttgtacacacc tgtacacacc gcccgtcgct gcccgtcgct 1620 1620 agtaccgatt gaatggctta agtaccgatt gaatggcttagtgaggcctc gtgaggcctcaggatctgct aggatctgct tagaagaggg tagaagaggg ggcgactcca ggcgactcca 1680 1680 cttcagagcg gagaatctgg tcaaacttgg tcatttagag gaaacccaaa aa cttcagagcg gagaatctgg tcaaacttgg tcatttagag gaaacccaaa aa 1732 1732
<210> <210> 39 39 <211> <211> 1223 1223 <212> <212> DNA DNA <213> <213> Candida parapsilosis Candida parapsilosis
<400> <400> 39 39 gcctgagaaa cggctaccacatccaaggaa gcctgagaaa cggctaccac atccaaggaaggcagcaggc ggcagcaggc gcgcaaatta gcgcaaatta cccaatcccg cccaatcccg
acacggggag gtagtgacaa acacggggag gtagtgacaataaataacga taaataacgatacagggccc tacagggccc tttcgggtct tttcgggtct tgtaattgga tgtaattgga 120 120
atgagtacaa tgtaaatacc atgagtacaa tgtaaataccttaacgagga ttaacgaggaacaattggag acaattggag ggcaagtctg ggcaaattctg gtgccagcag gtgccagcag 180 180
ccgcggtaat tccagctcca ccgcggtaat tccagctccaaaagcgtata aaagcgtatattaaagttgt ttaaagttgt tgcagttaaa tgcagttaaa aagctcgtag aagctcgtag 240 240
ttgaaccttg ggcttggctg ttgaaccttg ggcttggctggccggtccat gccggtccatcttttttgat cttttttgat gcgtactgga gcgtactgga cccagccgag cccagccgag 300 300
cctttccttc tggctagcct cctttccttc tggctagcctttttggcgaa ttttggcgaaccaggacttt ccaggacttt tactttgaaa tactttgaaa aaattagagt aaattagagt 360 gttcaaagca ggcctttgct gttcaaagca ggcctttgctcgaatatatt cgaatatattagcatggaat agcatggaat aatagaatag aatagaatag gacgttatgg gacgttatgg 420 420 ttctattttg ttggtttcta ggaccatcgt ttctattttg ttggtttcta ggaccatcgtaatgattaat aatgattaat agggacggtc agggacggtc gggggtatca gggggtatca 480 480 gtattcagta gtcagaggtg gtattcagta gtcagaggtgaaattcttgg aaattcttggatttactgaa atttactgaa gactaactac gactaactac tgcgaaagca tgcgaaagca 540 540 tttaccaagg acgttttcat taatcaagaa tttaccaagg acgttttcat taatcaagaacgaaagttag cgaaagttag gggatcgaag gggatcgaag atgatcagat atgatcagat 600 600 accgtcgtag tcttaaccat accgtcgtag tcttaaccataaactatgcc aaactatgccgactagggat gactagggat cggttgttgt cggttgttgt tcttttattg tcttttattg 660 660 acgcaatcgg caccttacga acgcaatcgg caccttacgagaaatcaaag gaaatcaaagtctttgggtt tctttgggtt ctggggggag ctggggggag tatggtcgca tatggtcgca 720 720 aaggctgaaa cttaaaggaa aaggctgaaa cttaaaggaattgacggaag ttgacggaagggcaccacca ggcaccacca ggagtggagc ggagtggage ctgcggctta ctgcggctta 780 780 atttgactca acacggggaa atttgactca acacggggaaactcaccagg actcaccaggtccagacaca tccagacaca ataaggattg ataaggattg acagattgag acagattgag 840 840 agctctttct tgattttgtg agctctttct tgattttgtgggtggtggtg ggtggtggtgcatggccgtt catggccgtt cttagttggt cttagttggt ggagtgattt ggagtgattt 900 900 gtctgcttaa ttgcgataac gtctgcttaa ttgcgataacgaacgagacc gaacgagaccttaacctact ttaacctact aaatagtgct aaatagtgct gctagcattt gctagcattt 960 960 gctggtatag tcacttctta gctggtatag tcacttcttagagggactat gagggactatcgatttcaag cgatttcaag tcgatggaag tcgatggaag tttgaggcaa tttgaggcaa 1020 1020 taacaggtct gtgatgccct tagacgttct taacaggtct gtgatgccct tagacgttctgggccgcacg gggccgcacg cgcgctacac cgcgctacac tgacggagcc tgacggagcc 1080 1080 agcgagtata aaccttggcc agcgagtata aaccttggccgagaggtctg gagaggtctgggaaatcttg ggaaatcttg tgaaactccg tgaaactccg tcgtgctggg tcgtgctggg 1140 1140 gatagagcat tgtaattatt gatagagcat tgtaattattgctcttcaac gctcttcaacgaggaattcc gaggaattcc tagtaagcgc tagtaagcgc aagtcatcag aagtcatcag 1200 1200 c t t g c g t t g a t t a c g t c c c t g c c g c C cttgcgttga 1223 1223 ttacgtccct
<210> <210> 40 40 <211> <211> 1563 1563 <212> <212> DNA DNA <213> <213> Candida tropicalis Candida tropicalis
<220> <220> <221> <221> misc_feature misc_feature <222> <222> (69)..(69) (69)..(69) <223> <223> n is a, n is a, C, c, g, g, or or t t
<220> <220> <221> misc_feature <221> misc_feature <222> <222> (627)..(627) (627)..(627) <223> n isa, <223> n is a,C,c,g, g,or ortt
<220> <220> <221> misc_feature <221> misc_feature <222> (631)..(631) <222> (631)..(631) <223> <223> nnis isa, a,C,c,g, g,or ortt
<220> <220> <221> misc_feature <221> misc_feature <222> <222> (674)..(674) (674)..(674) <223> n isa, <223> n is a,C,c,g, g,or ortt
<220> <220> <221> misc_feature <221> misc_feature <222> (684)..(684) <222> (684)..(684) <223> <223> nn is is a, a, c, c, g, g, or or t t
<220> <220> <221> misc_feature <221> misc_feature <222> <222> (699)..(699) (699)..(699) <223> n isa, <223> n is a,C,c,g, g,or ortt
<220> <220> <221> <221> misc_feature misc_feature <222> <222> (704)..(704) (704)..(704) <223> <223> n is n is a, a, C, c, g, g, or or t t
<220> <220> <221> misc_feature <221> misc_feature
<222> (723)..(822) <222> (723)..(822) <223> <223> nnis isa, a,C, c,g, g,or ortt
<220> <220> <221> misc_feature <221> misc feature <222> <222> (866)..(866) (866)..(866) <223> <223> nn is is a, a, C, c, g, g, or or tt
<220> <220> <221> misc_feature <221> misc_feature <222> <222> (1014)..(1014) (1014)..(1014) <223> n is a, <223> n is a, C, c,g, g,orort t
<220> <220> <221> misc_feature <221> misc_feature <222> <222> (1509)..(1509) (1509)..(1509) <223> n is a, <223> n is a, C, c, g, g, or or tt
<220> <220> <221> misc_feature <221> misc_feature <222> (1542)..(1542) <222> (1542)..(1542) <223> <223> n is n is a, a, C, c,g, g,orort t
<400> <400> 40 40 atgcttgtct caagattaagccatgcatgt atgcttgtct caagattaag ccatgcatgtctaagtataa ctaagtataa gcaatttata gcaatttata cagtgaaact cagtgaaact
gcgaatggnt cattaaatca gcgaatggnt cattaaatcagttatcgttt gttatcgtttatttgatagt atttgatagt accttactac accttactac ttggataacc ttggataacc 120 120
gtggtaattc tagagctaat gtggtaattc tagagctaatacatgcttaa acatgcttaaaatcccgact aatcccgact gtttggaagg gtttggaagg gatgtattta gatgtattta 180 180
ttagataaaa aatcaatgtcttcggactct ttagataaaa aatcaatgtc ttcggactctttgatgatto ttgatgattc ataataactt ataataactt ttcgaatcgc ttcgaatcgc 240 240
atggccttgt gctggcgatg atggccttgt gctggcgatggttcattcaa gttcattcaaatttctgccc atttctgccc tatcaacttt tatcaacttt cgatggtagg cgatggtagg 300 300
atagtggcct accatggtttcaacgggtaa atagtggcct accatggttt caacgggtaacggggaataa cggggaataa gggttcgatt gggttcgatt ccggagaggg ccggagaggg 360 360
agcctgagaa acggctacca agcctgagaa acggctaccacatccaagga catccaaggaaggcagcagg aggcagcagg cgcgcaaatt cgcgcaaatt acccaatccc acccaatccc 420 gacacgggga ggtagtgaca gacacgggga ggtagtgacaataaataacg ataaataacgatacagggcc atacagggcc ctttcgggtc ctttcgggtc ttgtaattgg ttgtaattgg 480 480 aatgagtaca atgtaaatac aatgagtaca atgtaaataccttaacgagg cttaacgaggaacaattgga aacaattgga gggcaagtct gggcaagtct ggtgccagca ggtgccagca 540 540 gcccgcggta attccagctt gcccgcggta attccagcttcaaaagccgt caaaagccgtatattaaagg atattaaagg tggttgcagt tggttgcagt taaaaagctc taaaaagctc 600 600 gtagttgaac cttgggcttt gtagttgaac cttgggctttggttggnccg ggttggnccgnccatctttc nccatctttc tgaagcctac tgaagectac tggaccccaa tggaccccaa 660 660 cccgagccct ttcntttggc cccgagccct ttcntttggctaancctttt taanccttttggcgaaccng ggcgaaccng gacntttacc gacntttacc tttgaaaaaa tttgaaaaaa 720 720 ttnnnnnnnn nnnnnnnnnnnnnnnnnnnn ttnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 780 780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nncgattagg nncgattagg gatcggttgt gatcggttgt 840 840 tgttctttaa ttgacgccat tgttctttaa ttgacgccattgggcncctt tgggcnccttacgagaaatc acgagaaatc aaaagtcttt aaaagtcttt gggttctggg gggttctggg 900 900 ggaagtatgg tcgcaaggtt ggaagtatgg tcgcaaggttgaaactttaa gaaactttaaaggaattgac aggaattgac ggaagggcac ggaagggcac caccaggagt caccaggagt 960 960 ggagcctgcg ggcttaattt ggagcctgcg ggcttaatttgactcaacac gactcaacacggggaaactc ggggaaactc accaggtcca accaggtcca gacncaataa gacncaataa 1020 1020 ggattgacag attgagagct ggattgacag attgagagctctttcttgat ctttcttgattttgtgggtg tttgtgggtg gtggtgcatg gtggtgcatg gccgttctta gccgttctta 1080 1080 gttggtggag tgatttgtct gttggtggag tgatttgtctgcttaattgc gcttaattgcgataacgaac gataacgaac gagaccttaa gagaccttaa cctactaaat cctactaaat 1140 1140 agtgctgcta gcatttgctg agtgctgcta gcatttgctggtatagtcac gtatagtcacttcttagagg ttcttagagg gactatcgat gactatcgat ttcaagtcga ttcaagtcga 1200 1200 tggaagtttg aggcaataac tggaagtttg aggcaataacaggtctgtga aggtctgtgatgcccttaga tgcccttaga cgttctgggc cgttctgggc cgcacgcgcg cgcacgcgcg 1260 1260 ctacactgac ggagccagcg ctacactgac ggagccagcgagtataaacc agtataaaccttggccgaga ttggccgaga ggtctgggaa ggtctgggaa atcttgtgaa atcttgtgaa 1320 actccgtcgt gctggggata actccgtcgt gctggggatagagcattgta gagcattgtaattgttgctc attgttgctc ttcaacgagg ttcaacgagg aattcctagt aattcctagt 1380 1380 aagcgcaagt catcagcttg aagcgcaagt catcagcttgcgttgattac cgttgattacgtccctgccc gtccctgccc tttgtacaca tttgtacaca ccgcccgtcg ccgcccgtcg 1440 1440 ctactaccga ttgaatggct ctactaccga ttgaatggcttagtgaggct tagtgaggcttccggattgg tccggattgg tttaggaaag tttaggaaag ggggcaactc ggggcaactc 1500 1500 cattctggna ccgagaagct cattctggna ccgagaagctagtcaaactc agtcaaactcggtcatttag ggtcatttag ancaagtaaa ancaagtaaa agtcgaacaa agtcgaacaa 1560 1560 g g g g t t 1563 1563
<210> <210> 41 41 <211> <211> 1801 1801 <212> <212> DNA DNA <213> <213> Cryptococcusneoformans Cryptococcus neoformans
<400> <400> 41 41 acctggttga tcctgccagtagtcatatgc acctggttga tcctgccagt agtcatatgcttgtctcaaa ttgtctcaaa gattaagcca gattaagcca tgcatgtcta tgcatgtcta
agtataaacg aattcatact agtataaacg aattcatactgtgaaactgc gtgaaactgcgaatggctca gaatggctca ttaaatcagt ttaaatcagt tatagtttat tatagtttat 120 120
ttgatggtat cttgctacat ttgatggtat cttgctacatggataactgt ggataactgtggtaattcta ggtaattcta gagctaatac gagctaatac atgctgaaaa atgctgaaaa 180 180
gccccgactt ctggaagggg gccccgactt ctggaaggggtgtatttatt tgtatttattagataaaaaa agataaaaaa ccaatgggtt ccaatgggtt tcggccctct tcggccctct 240 240
atggtgaatc ataataactt atggtgaatc ataataacttctcgaatcgc ctcgaatcgcatggccttgt atggccttgt gccggcgatg gccggcgatg cttcattcaa cttcattcaa 300 300
atatctgccc tatcaacttt atatctgccc tatcaactttcgatggtagg cgatggtaggatagaggect atagaggcct accatggtat accatggtat caacgggtaa caacgggtaa 360 360
cggggaatta gggttcgatt cggggaatta gggttcgattccggagaggg ccggagagggagcctgagaa agcctgagaa acggctacca acggctacca catccaagga catccaagga 420 420
aggcagcagg cgcgcaaatt aggcagcagg cgcgcaaattacccaatccc acccaatcccgacacgggga gacacgggga ggtagtgaca ggtagtgaca ataaataaca ataaataaca 480 atacagggct cttttgggcc atacagggct cttttgggccttgtaattgg ttgtaattggaatgagtaca aatgagtaca atttaaatcc atttaaatcc cttaacgagg cttaacgagg 540 540 aacaactgga gggcaagtct aacaactgga gggcaagtctggtgccagca ggtgccagcagccgcggtaa gccgcggtaa ttccagctcc ttccagctcc agtagcgtat agtagcgtat 600 600 attaaagttg ttgcagttaa attaaagttg ttgcagttaaaaagctcgta aaagctcgtagtcgaacttc gtcgaacttc aggtctggcg aggtctggcg aggcggtcct aggcggtcct 660 660 cctcacggag tgcactgtct cctcacggag tgcactgtcttgctggacct tgctggaccttacctcctgg tacctcctgg tggtcctgta tggtcctgta tgctctttac tgctctttac 720 720 tgggtgtgca ggggaaccag tgggtgtgca ggggaaccaggaattttacc gaattttaccttgaaaaaat ttgaaaaaat tagagtgttc tagagtgttc aaagcaggca aaagcaggca 780 780 atcgcccgaa tacattagca tggaataata atcgcccgaa tacattagca tggaataatagaataggacg gaataggacg tgcggttcta tgcggttcta ttttgttggt ttttgttggt 840 840 ttctaggatc gccgtaatga ttctaggatc gccgtaatgattaataggga ttaatagggacggtcggggg cggtcggggg cattggtatt cattggtatt ccgttgctag ccgttgctag 900 900 aggtgaaatt cttagattga aggtgaaatt cttagattgacggaagacca cggaagaccaacaactgcga acaactgcga aagcatttgc aagcatttgc caaggacgtt caaggacgtt 960 960 ttcattgatc aagaacgaag ttcattgate aagaacgaaggttaggggat gttaggggatcaaaaacgat caaaaacgat tagataccgt tagataccgt tgtagtctta tgtagtctta 1020 1020 acagtaaacg atgccgacta acagtaaacg atgccgactagggatcggcc gggatcggcccacgtcaate cacgtcaatc tctgactggg tctgactggg tcggcacctt tcggcacctt 1080 1080 acgagaaatc aaagtctttg acgagaaatc aaagtctttgggttctgggg ggttctggggggagtatggt ggagtatggt cgcaaggctg cgcaaggctg aaacttaaag aaacttaaag 1140 1140 gaattgacgg aagggcacca gaattgacgg aagggcaccaccaggtgtgg ccaggtgtggagcctgcggc agcctgcggc ttaatttgac ttaatttgac tcaacacggg tcaacacggg 1200 1200 gaaactcacc aggtccagac gaaactcacc aggtccagacatagtgagga atagtgaggattgacagatt ttgacagatt gatagctctt gatagctctt tcttgattct tcttgattct 1260 1260 atgggtggtg gtgcatggcc atgggtggtg gtgcatggccgttcttagtt gttcttagttggtggagtga ggtggagtga tttgtctggt tttgtctggt taattccgat taattccgat 1320 1320 aacgaacgag accttaacct aacgaacgag accttaacctgctaaatagt gctaaatagtcaggccggct caggccggct ttggctggtc ttggctggtc gtatgacttc gtatgacttc 1380 ttagagggac tgtcggcgtc tagtcgacgg ttagagggac tgtcggcgtc tagtcgacggaagtttgagg aagtttgagg caataacagg caataacagg tctgtgatgc tctgtgatgc 1440 1440 ccttagatgt tctgggccgc ccttagatgt tctgggccgcacgcgcgcta acgcgcgctacactgactga cactgactga gccagcgagt gccagcgagt cttaccgcct cttaccgcct 1500 1500 tggccgagag gcctgggtaa tggccgagag gcctgggtaatcttgtgaaa tcttgtgaaactcagtcgtg ctcagtcgtg ctggggatag ctggggatag agcattgcaa agcattgcaa 1560 1560 ttattgctct tcaacgagga atacctagta ttattgctct tcaacgagga atacctagtaagcgtgagtc agcgtgagtc accagctcgc accagctcgc gttgattacg gttgattacg 1620 1620 tccctgccct ttgtacacac cgcccgtcgc tccctgccct ttgtacacac cgcccgtcgctactaccgat tactaccgat tgaatggctt tgaatggctt agtgagatct agtgagatct 1680 1680 ccggattggc gttggggagc ccggattggc gttggggagccggcaaccgc cggcaacggcaccccttggc accccttggc cgagaagttg cgagaagttg atcaaacttg atcaaacttg 1740 1740 gtcatttaga ggaagtaaaa gtcatttaga ggaagtaaaagtcgtaacaa gtcgtaacaaggtttccgta ggtttccgta ggtgaacctg ggtgaacctg cggaaggatc cggaaggatc 1800 1800 a a 1801 1801
<210> <210> 42 42 <211> <211> 1673 1673 <212> <212> DNA DNA <213> <213> Fusarium sp. Fusarium sp.
<400> <400> 42 42 gcaattatac cgcgaaactgcgaatggctc gcaattatac cgcgaaactg cgaatggctcattatataag attatataag ttatcgttta ttatcgttta tttgatagta tttgatagta
ccttactact tggataaccg ccttactact tggataaccgtggtaattct tggtaattctagagctaata agagctaata catgctaaaa catgctaaaa atcccgactt atcccgactt 120 120
cggaagggat gtatttatta cggaagggat gtatttattagattaaaaac gattaaaaaccaatgccctt caatgccctt cggggctcac cggggctcac tggtgattca tggtgattca 180 180
tgataactcc tcgaatcgca tgataactcc tcgaatcgcatggccttgtg tggccttgtgccggcgatgg ccggcgatgg ttcattcaaa ttcattcaaa tttcttccct tttcttccct 240 240
atcaactttc gatgtttgggtattggccaa atcaactttc gatgtttggg tattggccaaacatggttgc acatggttgc aacgggtaac aacgggtaac ggagggttag ggagggttag 300 ggctcgaccc cggagaagga ggctcgaccc cggagaaggagcctgagaaa gcctgagaaacggctactac cggctactac atccaaggaa atccaaggaa ggcagcaggc ggcagcaggc 360 360 gcgcaaatta cccaatcccg gcgcaaatta cccaatcccgacacggggag acacggggaggtagtgacaa gtagtgacaa taaatactga taaatactga tacagggctc tacagggctc 420 420 ttttgggtct tgtaattggaatgagtacaa ttttgggtct tgtaattgga atgagtacaatttaaatccc tttaaatccc ttaacgagga ttaacgagga acaattggag acaattggag 480 480 ggcaagtctg gtgccagcag ggcaagtctg gtgccagcagccgcggtaat ccgcggtaattccagctcca tccagctcca atagcgtata atagcgtata ttaaagttgt ttaaagttgt 540 540 tgtggttaaa aagctcgtag tgtggttaaa aagctcgtagttgaaccttg ttgaaccttgggcctggccg ggcctggccg tccggtccgc tccggtccgc ctcaccgcgt ctcaccgcgt 600 600 gtactggctc ggccgggcct gtactggctc ggccgggcctttccctctgt ttccctctgtggaaccccat ggaaccccat gcccttcact gcccttcact gggcgtggcg gggcgtggcg 660 660 gggaaacagg acttttactg gggaaacagg acttttactgtgaaaaaatt tgaaaaaattagagtgctcc agagtgctcc aggcaggcct aggcaggcct atgctcgaat atgctcgaat 720 720 acattagcat ggaataatag acattagcat ggaataatagaataggacgt aataggacgtgtggttctat gtggttctat tttgttggtt tttgttggtt tctaggaccg tctaggaccg 780 780 ccgtaatgat taatagggac ccgtaatgat taatagggacagtcgggggc agtcgggggcatcagtattc atcagtattc aattgtcaga aattgtcaga ggtgaaattc ggtgaaattc 840 840 ttggatttat tgaagactaactactgcgaa ttggatttat tgaagactaa ctactgcgaaagcatttgcc agcatttgcc aaggatgttt aaggatgttt tcattaatca tcattaatca 900 900 ggaacgaaag ttaggggatc ggaacgaaag ttaggggatcgaagacgatc gaagacgatcagataccgtc agataccgtc gtagtcttaa gtagtcttaa ccataaacta ccataaacta 960 960 tgccgactag ggatcggacggtgttatttt tgccgactag ggatcggacg gtgttattttttgacccgtt ttgacccgtt cggcacctta cggcacctta cgagaaatca cgagaaatca 1020 1020 aagtgcttgg gctccagggg aagtgcttgg gctccagggggagtatggtc gagtatggtcgcaaggctga gcaaggctga aacttaaaga aacttaaaga aattgacgga aattgacgga 1080 1080 agggcaccac caggggtgga agggcaccac caggggtggagcctgcggct gcctgcggcttaatttgact taatttgact caacacgggg caacacgggg aaactcacca aaactcacca 1140 1140 ggtccagaca caatgaggat ggtccagaca caatgaggattgacagattg tgacagattgagagctcttt agagctcttt cttgattttg cttgattttg tgggtggtgg tgggtggtgg 1200 tgcatggccg ttcttagttg tgcatggccg ttcttagttggtggagtgat gtggagtgatttgtctgctt ttgtctgctt aattgcgata aattgcgata acgaacgaga acgaacgaga 1260 1260 ccttaacctg ctaaatagcc ccttaacctg ctaaatagcccgtattgctt cgtattgctttggcagtacg tggcagtacg ctggcttctt ctggcttctt agagggacta agagggacta 1320 1320 tcggctcaag ccgatggaag tcggctcaag ccgatggaagtttgaggcaa tttgaggcaataacaggtct taacaggtct gtgatgccct gtgatgccct tagatgttct tagatgttct 1380 1380 gggccgcacg cgcgctacac gggccgcacg cgcgctacactgacggagcc tgacggagccagcgagtact agcgagtact tccttgtccg tccttgtccg aaaggtccgg aaaggtccgg 1440 1440 gtaatcttgt taaactccgt gtaatcttgt taaactccgtcgtgctgggg cgtgctggggatagagcatt atagagcatt gcaattattg gcaattattg ctcttcaacg ctcttcaacg 1500 1500 aggaatccct agtaagcgca aggaatccct agtaagcgcaagtcatcago agtcatcagcttgcgttgat ttgcgttgat tacgtccctg tacgtccctg ccctttgtac ccctttgtac 1560 1560 acaccgcccg tcgctactac acaccgcccg tcgctactaccgattgaatg cgattgaatggctcagtgag gctcagtgag gcgtccggac gcgtccggac tggcccagag tggcccagag 1620 1620 aggtgggcaa ctaccactca gggccggaaa gctctccaaa ctcggtcatt aga aggtgggcaa ctaccactca gggccggaaa gctctccaaa ctcggtcatt aga 1673 1673
<210> <210> 43 43 <211> <211> 1554 1554 <212> <212> DNA DNA <213> <213> Bacillus anthracis Bacillus anthracis
<400> <400> 43 43 ttattggaga gtttgatcctggctcaggat ttattggaga gtttgatcct ggctcaggatgaacgctggc gaacgctggc ggcgtgccta ggcgtgccta atacatgcaa atacatgcaa
gtcgagcgaa tggattaaga gtcgagcgaa tggattaagagcttgctctt gcttgctcttatgaagttag atgaagttag cggcggacgg cggcggacgg gtgagtaaca gtgagtaaca 120 120
cgtgggtaac ctgcccataa cgtgggtaac ctgcccataagactgggata gactgggataactccgggaa actccgggaa accggggcta accggggcta ataccggata ataccggata 180 180
acattttgaa ccgcatggtt acattttgaa ccgcatggttcgaaattgaa cgaaattgaaaggcggcttc aggcggcttc ggctgtcact ggctgtcact tatggatgga tatggatgga 240 240
cccgcgtcgc attagctagt cccgcgtcgc attagctagttggtgaggta tggtgaggtaacggctcacc acggctcacc aaggcaacga aaggcaacga tgcgtagccg tgcgtagccg 300 acctgagagg gtgatcggcc acctgagagg gtgatcggccacactgggac acactgggactgagacacgg tgagacacgg cccagactcc cccagactcc tacgggaggc tacgggaggc 360 360 agcagtaggg aatcttccgc agcagtaggg aatcttccgcaatggacgaa aatggacgaaagtctgacgg agtctgacgg agcaacgccg agcaaccccg cgtgagtgat cgtgagtgat 420 420 gaaggctttc gggtcgtaaa gaaggctttc gggtcgtaaaactctgttgt actctgttgttagggaagaa tagggaagaa caagtgctag caagtgctag ttgaataagc ttgaataage 480 480 tggcaccttg acggtaccta tggcaccttg acggtacctaaccagaaage accagaaagccacggctaac cacggctaac tacgtgccag tacgtgccag cagccgcggt cagccgcggt 540 540 aatacgtagg tggcaagcgt aatacgtagg tggcaagcgttatccggaat tatccggaattattgggcgt tattgggcgt aaagcgcgcg aaagcgcgcg caggtggttt caggtggttt 600 600 cttaagtctg atgtgaaagcccacggctca cttaagtctg atgtgaaago ccacggctcaaccgtggagg accgtggagg gtcattggaa gtcattggaa actgggagac actgggagac 660 660 ttgagtgcag aagaggaaag ttgagtgcag aagaggaaagtggaattcca tggaattccatgtgtagcgg tgtgtagcgg tgaaatgcgt tgaaatgcgt agagatatgg agagatatgg 720 720 aggaacacca gtggcgaagg aggaacacca gtggcgaaggcgactttctg cgactttctggtctgtaact gtctgtaact gacactgagg gacactgagg cgcgaaagcg cgcgaaagcg 780 780 tggggagcaa acaggattag tggggagcaa acaggattagataccctggt ataccctggtagtccacgcc agtccacgcc gtaaacgatg gtaaacgatg agtgctaagt agtgctaagt 840 840 gttagagggt ttccgccctt gttagagggt ttccgccctttagtgctgaa tagtgctgaagttaacgcat gttaacgcat taagcactcc taagcactcc gcctggggag gcctggggag 900 900 tacggccgca aggctgaaac tacggccgca aggctgaaactcaaaggaat tcaaaggaattgacgggggc tgacgggggc ccgcacaagc ccgcacaage ggtggagcat ggtggagcat 960 960 gtggtttaat tcgaagcaac gtggtttaat tcgaagcaacgcgaagaacc gcgaagaaccttaccaggtc ttaccaggtc ttgacatcct ttgacatcct ctgacaaccc ctgacaaccc 1020 1020 tagagatagg gcttctcctt tagagatagg gcttctccttcgggagcaga cgggagcagagtgacaggtg gtgacaggtg gtgcatggtt gtgcatggtt gtcgtcagct gtcgtcagct 1080 1080 cgtgtcgtga gatgttgggt cgtgtcgtga gatgttgggttaagtcccgc taagtcccgcaacgagcgca aacgagcgca acccttgatc acccttgatc ttagttgcca ttagttgcca 1140 1140 tcattwagtt gggcactcta aggtgactgc tcattwagtt gggcactcta aggtgactgccggtgacaaa cggtgacaaa ccggaggaag ccggaggaag gtggggatga gtggggatga 1200 cgtcaaatca tcatgcccct cgtcaaatca tcatgccccttatgacctgg tatgacctgggctacacacg gctacacacg tgctacaatg tgctacaatg gacggtacaa gacggtacaa 1260 1260 agagctgcaa gaccgcgagg agagctgcaa gaccgcgaggtggagctaat tggagctaatctcataaaac ctcataaaac cgttctcagt cgttctcagt tcggattgta tcggattgta 1320 1320 ggctgcaact cgcctacatg ggctgcaact cgcctacatgaagctggaat aagctggaatcgctagtaat cgctagtaat cgcggatcag cgcggatcag catgccgcgg catgccgcgg 1380 1380 tgaatacgtt cccgggcctt tgaatacgtt cccgggccttgtacacaccg gtacacaccgcccgtcacac cccgtcacac cacgagagtt cacgagagtt tgtaacaccc tgtaacaccc 1440 1440 gaagtcggtg gggtaacctt gaagtcggtg gggtaacctttttggagcca tttggagccagccgcctaag gccgcctaag gtgggacaga gtgggacaga tgattggggt tgattggggt 1500 1500 gaagtcgtaa caaggtagcc gaagtcgtaa caaggtagcc gtatcggaag gtatcggaag gtgcggctgg gtgcggctgg atcacctcct atcacctcct ttct ttct 1554 1554
<210> <210> 44 44 <211> <211> 1610 1610 <212> <212> DNA DNA <213> <213> Burkholderiapseudomallei Burkholderia pseudomallei
<400> <400> 44 44 tctagatgcg tgctcgagcggccgcccagt tctagatgcg tgctcgagcg gccgcccagtgctgcatgga gctgcatgga tatctgctga tatctgctga attcggcttg attcggcttg
agcagtttga tcctggctca agcagtttga tcctggctcagattgaacgc gattgaacgctggcggcatg tggcggcatg ccttacacat ccttacacat gcaagtcgaa gcaagtcgaa 120 120
cggcagcacg ggcttcggcc cggcagcacg ggcttcggcctggtggcgag tggtggcgagtggcgaacgg tggcgaacgg gtgagttata gtgagttata catcggagca catcggagca 180 180
tgtcctgtag tgggggatag tgtcctgtag tgggggatagcccggcgaaa cccggcgaaagccgaattaa gccgaattaa taccgcatac taccgcatac gatctgagga gatctgagga 240 240
tgaaagcggg ggaccttcgg tgaaagcggg ggaccttcgggcctcgcgct gcctcgcgctatagggttgg atagggttgg ccgatggctg ccgatggctg attagctagt attagctagt 300 300
tggtggggta aaggcctacc tggtggggta aaggcctaccaaggcgacga aaggcgacgatcagtagctg tcagtagctg gtctgagagg gtctgagagg acgaccagcc acgaccagcc 360 360
acactgggac tgagacacgg acactgggac tgagacacggcccagactcc cccagactcctacgggaggc tacgggaggc agcagtgggg agcagtgggg aattttggac aattttggac 420 aatgggcgca agcctgatcc aatgggcgca agcctgatccagcaatgccg agcaatgccgcgtgtgtgaa cgtgtgtgaa gaaggccttc gaaggccttc gggttgtaaa gggttgtaaa 480 480 gcacttttgt ccggaaagaa gcacttttgt ccggaaagaaatcattctgg atcattctggctaatacccg ctaatacccg gagtggatga gagtggatga cggtaccgga cggtaccgga 540 540 agaataagca ccggctaact agaataagca ccggctaactacgtgccage acgtgccagcagccgcggta agccgcggta atacgtaggg atacgtaggg tgcgagcgtt tgcgagcgtt 600 600 aatcgggatt actgggcgta aatcgggatt actgggcgtaaagcgtgcgc aagcgtgcgcaggcggtttg aggcggtttg ctaagaccga ctaagaccga tgtgaaatcc tgtgaaatcc 660 660 ccgggctcaa cctgggaact ccgggctcaa cctgggaactgcattggtga gcattggtgactggcaggct ctggcaggct agagtatggc agagtatggc agaggggggt agaggggggt 720 720 agaattccac gtgtagcagt agaattccac gtgtagcagtgaaatgcgta gaaatgcgtagagatgtgga gagatgtgga ggaataccga ggaataccga tggcgaaggc tggcgaaggc 780 780 agccccctgg gccaatactg agccccctgg gccaatactgacgctcatgc acgctcatgcacgaaagcgt acgaaagcgt ggggagaaaa ggggagaaaa caggattaga caggattaga 840 840 taccctggta gtccacgccctaaacgatgt taccctggta gtccacgccc taaacgatgtcaactagttg caactagttg ttggggattc ttggggattc atttccttag atttccttag 900 900 taacgtagct aacgcgcgaa taacgtagct aacgcgcgaagttgaccgcc gttgaccgcctggggagtac tggggagtac ggtcgcaaga ggtcgcaaga ttaaaactca ttaaaactca 960 960 aaggaattga cggggacccg cacaagcggt aaggaattga cggggacccg cacaagcggtggatgatgtg ggatgatgtg gattaattcg gattaattcg atgcaacgcg atgcaacccg 1020 1020 aaaaacctta cctacccttg aaaaacctta cctacccttgacatggtcgg acatggtcggaagcccgatg aagcccgatg agagttgggc agagttgggc gtgctcgaaa gtgctcgaaa 1080 1080 gagaaccggc gcacaggtgc gagaaccggc gcacaggtgctgcatggctg tgcatggctgtcgtcagctc tcgtcagctc gtgtcgtgag gtgtcgtgag atgttgggtt atgttgggtt 1140 1140 aagtcccgca acgagcgcaa aagtcccgca acgagcgcaacccttgtcct cccttgtccttagttgctac tagttgctac gcaagagcac gcaagagcac tctaaggaga tctaaggaga 1200 1200 ctgccggtga caaaccggaggaaggtgggg ctgccggtga caaaccggag gaaggtggggatgacgtcaa atgacgtcaa gtcctcatgg gtcctcatgg cccttatggg cccttatggg 1260 1260 tagggcttca cacgtcatac aatggtcgga tagggcttca cacgtcatac aatggtcggaacagagggtc acagagggtc gccaacccgc gccaacccgc gagggggagc gagggggage 1320 caatcccaga aaaccgatcg caatcccaga aaaccgatcgtagtccggat tagtccggattgcactctgc tgcactctgc aactcgagtg aactcgagtg catgaagctg catgaagctg 1380 1380 gaatcgctag taatcgcgga gaatcgctag taatcgcggatcagcatgcc tcagcatgccgcggtgaata gcggtgaata cgttcccggg cgttcccggg tcttgtacac tcttgtacac 1440 1440 accgcccgtc acaccatggg accgcccgtc acaccatgggagtgggtttt agtgggttttaccagaagtg accagaagtg gctagtctaa gctagtctaa ccgcaaggag ccgcaaggag 1500 1500 gacggtcacc acggtaggat gacggtcacc acggtaggattcatgactgg tcatgactggggtgaagtcg ggtgaagtcg taacaaggta taacaaggta gccgtagaag gccgtagaag 1560 1560 ccgaattcca gcacactggc ggccgttact actggatccg agctcgtacc ccgaattcca 1610 gcacactggc ggccgttact actggatccg agctcgtacc 1610
<210> <210> 45 45 <211> <211> 1511 1511 <212> <212> DNA DNA <213> <213> Clostridium botulinum Clostridium botulinum
<220> <220> <221> misc_feature <221> misc_feature <222> <222> (1)..(7) (1)..(7) <223> <223> nnis isa, a,C,c,g, g,or ortt
<400> <400> 45 45 nnnnnnngag agtttgatcc nnnnnnngag agtttgatcctggctcagga tggctcaggacgaacgctgg cgaacgctgg cggcgtgctt cggcgtgctt aacacatgca aacacatgca
agtcgagcga tgaagcttcc agtcgagcga tgaagcttccttcgggaagt ttcgggaagtggattagcgg ggattagcgg cggacgggtg cggacgggtg agtaacacgt agtaacacgt 120 120
gggtaacctg cctcaaagtg gggtaacctg cctcaaagtgggggatagcc ggggatagccttccgaaagg ttccgaaagg aagattaata aagattaata ccgcataata ccgcataata 180 180
taagagaatc gcatgattttcttatcaaag taagagaatc gcatgatttt cttatcaaagatttattgct atttattgct ttgagatgga ttgagatgga cccgcggcgc cccgcggcgc 240 240
attagctagt tggtaaggta attagctagt tggtaaggtaacggcttacc acggcttaccaaggcaacga aaggcaacga tgcgtagccg tgcgtagccg acctgagagg acctgagagg 300 300
gtgatcggcc acattggaac gtgatcggcc acattggaactgagacacgg tgagacacggtccagactcc tccagactcc tacgggaggc tacgggaggc agcagtgggg agcagtgggg 360 aatattgcgc aatgggggag aatattgcgc aatgggggagaccctgacgc accctgacgcagcaaccccg agcaacgccg cgtgggtgat cgtgggtgat gaaggtcttc gaaggtcttc 420 420 ggattgtaaa gccctgtttt ggattgtaaa gccctgttttctaggacgat ctaggacgataatgacggta aatgacggta ctagaggagg ctagaggagg aagccacggc aagccacggc 480 480 taactacgtg ccagcagccg taactacgtg ccagcagccgcggtaatacg cggtaatacgtaggtggcga taggtggcga gcgttgtccg gcgttgtccg gatttactgg gatttactgg 540 540 gcgtaaaggg tgcgtaggcg gcgtaaaggg tgcgtaggcggatgtttaag gatgtttaagtgggatgtga tgggatgtga aatccccggg aatccccggg cttaacctgg cttaacctgg 600 600 gggctgcatt ccaaactgga gggctgcatt ccaaactggatatctagagt tatctagagtgcaggagagg gcaggagagg aaagcggaat aaagcggaat tcctagtgta tcctagtgta 660 660 gcggtgaaat gcgtagagat gcggtgaaat gcgtagagattaggaagaac taggaagaacaccagtggcg accagtggcg aaggcggctt aaggcggctt tctggactgt tctggactgt 720 720 aactgacgct gaggcacgaa aactgacgct gaggcacgaaagcgtgggta agcgtgggtagcaaacagga gcaaacagga ttagataccc ttagataccc tggtagtcca tggtagtcca 780 780 cgccgtaaac gatggatact cgccgtaaac gatggatactaggtgtaggg aggtgtagggggtatcaact ggtatcaact ccccctgtgc ccccctgtgc cgcagttaac cgcagttaac 840 840 acaataagta tcccgcctgg acaataagta tcccgcctggggagtacggt ggagtacggtcgcaagatta cgcaagatta aaactcaaag aaactcaaag gaattgacgg gaattgacgg 900 900 gggcccgcac aagcagcggagcatgtggtt gggcccgcac aagcagcgga gcatgtggtttaattcgaag taattcgaag caacgcgaag caacgcgaag aaccttacct aaccttacct 960 960 ggacttgaca tcccttgcat ggacttgaca tcccttgcatagcctagaga agcctagagataggtgaage taggtgaagc ccttcggggc ccttcggggc aaggagacag aaggagacag 1020 1020 gtggtgcatg gttgtcgtca gtggtgcatg gttgtcgtcagctcgtgtcg gctcgtgtcgtgagatgtta tgagatgtta ggttaagtcc ggttaagtcc tgcaacgagc tgcaaccage 1080 1080 gcaacccttg ttattagttg gcaacccttg ttattagttgctaccattaa ctaccattaagttgagcact gttgagcact ctaatgagac ctaatgagac tgcctgggta tgcctgggta 1140 1140 accaggagga aggtggggat accaggagga aggtggggatgacgtcaaat gacgtcaaatcatcatgccc catcatgccc cttatgtcca cttatgtcca gggctacaca gggctacaca 1200 1200 cgtgctacaa tggtaggtac cgtgctacaa tggtaggtacaataagacgc aataagacgcaagaccgtga aagaccgtga ggtggagcaa ggtggagcaa aacttataaa aacttataaa 1260 acctatctca gttcggattg acctatctca gttcggattgtaggctgcaa taggctgcaactcgcctaca ctcgcctaca tgaagctgga tgaagctgga gttgctagta gttgctagta 1320 1320 atcgcgaatc agaatgtcgc atcgcgaatc agaatgtcgcggtgaatacg ggtgaatacgttcccgggcc ttcccgggcc ttgtacacac ttgtacacac cgccccgtca cgccccgtca 1380 1380 caccatgaga gctggtaaca cccgaagtcc caccatgaga gctggtaaca cccgaagtccgtgaggtaac gtgaggtaac cgtaaggagc cgtaaggage cagcggccga cagcggccga 1440 1440 aggtgggatt agtgattggg aggtgggatt agtgattggggtgaagtcgt gtgaagtcgtaacaaggtag aacaaggtag ccgtaggaga ccgtaggaga acctgcggct acctgcggct 1500 1500 g g a t c a c c t c c C g g a t C a C C t C 1511 1511
<210> <210> 46 46 <211> <211> 1510 1510 <212> <212> DNA DNA <213> <213> Clostridium botulinum Clostridium botulinum
<220> <220> <221> misc_feature <221> misc_feature <222> <222> (1)..(7) (1)..(7) <223> n is <223> n is a, a, C, c,g,g,orort t
<400> <400> 46 46 nnnnnnngag agtttgatcc nnnnnnngag agtttgatcctggctcagga tggctcaggacgaacgctgg cgaacgctgg cggcgtgctt cggcgtgctt aacacatgca aacacatgca
agtcgagcga tgaagcttcc agtcgagcga tgaagcttccttcgggaagt ttcgggaagtggattagcgg ggattagcgg cggacgggtg cggacgggtg agtaacacgt agtaacacgt 120 120
gggtaacctg cctcaaagtg gggtaacctg cctcaaagtgggggatagcc ggggatagccttccgaaagg ttccgaaagg aagattaata aagattaata ccgcataaca ccgcataaca 180 180
taagagaatc gcatgattttcttatcaaag taagagaatc gcatgatttt cttatcaaagatttattgct atttattgct ttgagatgga ttgagatgga cccgcggcgc cccgcggcgc 240 240
attagctagt tggtaaggtaacggcttacc attagctagt tggtaaggta acggcttaccaaggcaacga aaggcaacga tgcgtagccg tgcgtagccg acctgagagg acctgagagg 300 300
gtgatcggcc acattggaac gtgatcggcc acattggaactgagacacgg tgagacacggtccagactcc tccagactcc tacgggaggc tacgggaggc aggagtgggg aggagtgggg 360 aatattgcgc aatgggggaa aatattgcgc aatgggggaaaccctgacgc accctgacgcagcaaccccg agcaacgccg cgtgggtgat cgtgggtgat gaaggtcttc gaaggtcttc 420 420 ggattgtaaa gccctgtttt ggattgtaaa gccctgttttctaggacgat ctaggacgataatgacggta aatgacggta ctagaggagg ctagaggagg aagccacggc aagccacggc 480 480 taactacgtg ccagcagccg taactacgtg ccagcagccgcggtaatacg cggtaatacgtaggtggcga taggtggcga gcgttgtccg gcgttgtccg gatttactgg gatttactgg 540 540 gcgtaaaggg tgcgtaggcg gcgtaaaggg tgcgtaggcggatgtttaag gatgtttaagtgggatgtga tgggatgtga aatccccggg aatccccggg cttaacctgg cttaacctgg 600 600 gggctgcatt ccaaactgga gggctgcatt ccaaactggatatctagagt tatctagagtgcaggagagg gcaggagagg aaagcggaat aaagcggaat tcctagtgta tcctagtgta 660 660 gcggtgaaat gcgtagagat gcggtgaaat gcgtagagattaggaagaac taggaagaacaccagtggcg accagtggcg aaggcggctt aaggcggctt tctggactgt tctggactgt 720 720 aactgacgct gaggcacgaa aactgacgct gaggcacgaaagcgtgggta agcgtgggtagcaaacagga gcaaacagga ttagataccc ttagataccc tggtagtcca tggtagtcca 780 780 cgccgtaaac gatggatact cgccgtaaac gatggatactaggtgtaggg aggtgtagggggtatcaact ggtatcaact ccccctgtgc ccccctgtgc cgcagttaac cgcagttaac 840 840 acaataagta tcccgcctgg acaataagta tcccgcctggggagtacggt ggagtacggtcgcaagatta cgcaagatta aaactcaaag aaactcaaag gaattgacgg gaattgacgg 900 900 gggcccgcac aagcagcgga gggcccgcac aagcagcggagcatgtggtt gcatgtggtttaattcgaag taattcgaag caacgcgaag caacgcgaag aaccttacct aaccttacct 960 960 ggacttgaca tcccttgcat ggacttgaca tcccttgcatagcctagaga agcctagagataggtgaage taggtgaagc ccttcggggc ccttcggggc aaggagacag aaggagacag 1020 1020 gtggtgcatg gttgtcgtca gtggtgcatg gttgtcgtcagctcgtgtcg gctcgtgtcgtgagatgtta tgagatgtta ggttaagtcc ggttaagtcc tgcaacgagc tgcaaccage 1080 1080 gcaacccttg ttattagttg gcaacccttg ttattagttgctaccattaa ctaccattaagttgagcact gttgagcact ctaatgagac ctaatgagac tgcctgggta tgcctgggta 1140 1140 accaggagga aggtggggat accaggagga aggtggggatgacgtcaaat gacgtcaaatcatcatgccc catcatgccc cttatgtcca cttatgtcca gggctacaca gggctacaca 1200 1200 cgtgctacaa tggtaggtac cgtgctacaa tggtaggtacaataagacgc aataagacgcaagaccgtga aagaccgtga ggtggagcaa ggtggagcaa aacttataaa aacttataaa 1260 acctatctca gttcggattg acctatctca gttcggattgtaggctgcaa taggctgcaactcgcctaca ctcgcctaca tgaagctgga tgaagctgga gttgctagta gttgctagta 1320 1320 atcgcgaatc agaatgtcgc atcgcgaatc agaatgtcgcggtgaatacg ggtgaatacgttcccgggcc ttcccgggcc ttgtacacac ttgtacacac cgcccgtcac cgcccgtcac 1380 1380 accatgagag ctggtaacac accatgagag ctggtaacacccgaagtccg ccgaagtccgtgaggtaacc tgaggtaacc gtaaggagcc gtaaggagcc agcggccgaa agcggccgaa 1440 1440 ggtgggatta gtgattgggg ggtgggatta gtgattggggtgaagtcgta tgaagtcgtaacaaggtage acaaggtagc cgtaggagaa cgtaggagaa cctgcggctg cctgcggctg 1500 1500 g g a a t t c C a a c C c C t t c C c C 1510 1510
<210> <210> 47 47 <211> <211> 1516 1516 <212> <212> DNA DNA <213> <213> Clostridium botulinum Clostridium botulinum
<220> <220> <221> <221> misc_feature misc_feature <222> <222> (1)..(7) (1)..(7) <223> <223> n is n is a, a, C, c, g, g, or or t t
<400> <400> 47 47 nnnnnnngag agtttgatcc nnnnnnngag agtttgatcctggctcagga tggctcaggacgaacgtggc cgaacgtggc ggcgtgccta ggcgtgccta acacatgcaa acacatgcaa
gtcgagcgat gaagcttcct gtcgagcgat gaagcttccttcggggagtg tcggggagtggattagcggc gattagcggc ggacgggtga ggacgggtga gtaacacgtg gtaacacgtg 120 120
ggtaacctgc ctcaaagagg ggtaacctgc ctcaaagagggggatagcct gggatagcctcccgaaaggg cccgaaaggg agattaatac agattaatac cgcataacat cgcataacat 180 180
tattttatgg catcatagaa tattttatgg catcatagaataatcaaagg taatcaaaggagcaatccgc agcaatccgc tttgattatg tttgattatg gacccgcgtc gacccgcgtc 240 240
gcattagcta gttggtgagg gcattagcta gttggtgaggtaacggctca taacggctcaccaaggcaac ccaaggcaac gatgcgtagc gatgcgtagc cgacctgaga cgacctgaga 300 300
gggtgatcgg ccacattgga gggtgatcgg ccacattggaactgagacac actgagacacggtccagact ggtccagact cctacgggag cctacgggag gcagcagtgg gcagcagtgg 360 ggaatattgc gcaatggggg ggaatattgc gcaatgggggaaaccctgac aaaccctgacgcagcaacgc gcagcaacgc cgcgtgagtg cgcgtgagtg atgaaggttt atgaaggttt 420 420 tcggatcgta aaactctgtc tcggatcgta aaactctgtctttagggacg tttagggacgataatgacgg ataatgacgg tacctaagga tacctaagga ggaagccacg ggaagccacg 480 480 gctaactacg tgccagcage gctaactacg tgccagcagccgcggtaata cgcggtaatacgtaggtggc cgtaggtggc aagcgttgtc aagcgttgtc cggatttact cggatttact 540 540 gggcgtaaag agtatgtagg gggcgtaaag agtatgtaggtgggtgctta tgggtgcttaagtcagatgt agtcagatgt gaaattcccg gaaattcccg ggcttaacct ggcttaacct 600 600 gggcgctgca tttgaaactg gggcgctgca tttgaaactgggcatctaga ggcatctagagtgcaggaga gtgcaggaga ggaaagtgga ggaaagtgga attcctagtg attcctagtg 660 660 tagcggtgaa atgcgtagag attaggaaga tagcggtgaa atgcgtagag attaggaagaacaccagtgg acaccagtgg cgaaggcgac cgaaggcgac tttctggact tttctggact 720 720 gtaactgaca ctgagatacg gtaactgaca ctgagatacgaaagcgtggg aaagcgtgggtagcaaacag tagcaaacag gattagatcc gattagatcc ccctggtagt ccctggtagt 780 780 ccacgccgta aacgatgaat ccacgccgta aacgatgaatactaggtgtc actaggtgtcggggggtacc ggggggtacc accctcggtg accctcggtg ccgcagcaaa ccgcagcaaa 840 840 cgcattaagt attccgcctg cgcattaagt attccgcctggggagtacgg gggagtacggtcgcaagatt tcgcaagatt aaaactcaaa aaaactcaaa ggaattgacg ggaattgacg 900 900 gggacccgca caagcagcgg gggacccgca caagcagcggagcatgtggt agcatgtggtttaattcgaa ttaattcgaa gcaacgcgaa gcaacgcgaa gaaccttacc gaaccttacc 960 960 tagacttgac atctcctgaa tagacttgac atctcctgaattactcttaa ttactcttaatcgaggaagt tcgaggaagt cccttcgggg cccttcgggg gacaggaaga gacaggaaga 1020 1020 caggtggtgc atggttgtcgtcagctcgtg caggtggtgc atggttgtcg tcagctcgtgtcgtgagatg tcgtgagatg ttgggttaag ttgggttaag tcccgcaacg tcccgcaacg 1080 1080 agcgcaaccc ttattgttag agcgcaaccc ttattgttagttgctactat ttgctactattaagttaage taagttaagc actctaacga actctaacga gactgccgcg gactgccgcg 1140 1140 gttaacgtag aggaaggtgg gttaacgtag aggaaggtggggatgacgtc ggatgacgtcaaatcatcat aaatcatcat gccccttatg gccccttatg tctagggcta tctagggcta 1200 1200 cacacgtgct acaatggctg cacacgtgct acaatggctggtacaaccag gtacaacgagcagcaaaccc cagcaaaccc gcgaggggga gcgaggggga gcaaaacttg gcaaaacttg 1260 aaagccagtc ccagttcgga aaagccagtc ccagttcggattgtaggctg ttgtaggctgaaactcgcct aaactcgcct acatgaagtt acatgaagtt ggagttgcta ggagttgcta 1320 1320 gtaatcgcgg aatcagcatg gtaatcgcgg aatcagcatgtcgcggtgaa tcgcggtgaatacgtccccg tacgtccccg ggtcttgtac ggtcttgtac acaccgcccg acaccgcccg 1380 1380 tcacaccatg agagccggta acacccgaag tcacaccatg agagccggta acacccgaagcccgtgaggt cccgtgaggt aaccgtaagg aaccgtaagg agccagcggt agccagcggt 1440 1440 cgaaggtggg attggtgatt cgaaggtggg attggtgattggggtgaagt ggggtgaagtcgtaacaagg cgtaacaagg tagccgtagg tagccgtagg agaacctgcg agaacctgcg 1500 1500 g t t g g a t c a c c t c c t t gttggatcac 1516 1516 ctcctt <210> <210> 48 48 <211> <211> 1516 1516 <212> <212> DNA DNA <213> <213> Clostridium botulinum Clostridium botulinum
<220> <220> <221> misc_feature <221> misc_feature <222> (1)..(7) <222> (1)..(7) <223> <223> nnis isa, a,C,c,g, g,or ortt
<220> <220> <221> misc_feature <221> misc_feature <222> <222> (373)..(373) (373)..(373) <223> n isa, <223> n is a,c,c,g, g,or ortt
<220> <220> <221> misc_feature <221> misc_feature <222> <222> (789)..(790) (789)..(790) <223> <223> nn is is a, a, C, c, g, g, or or tt
<400> <400> 48 48 nnnnnnngag agtttgatcctggctcagga nnnnnnngag agtttgatcc tggctcaggacgaacgctgg cgaacgctgg cggcgtgcct cggcgtgcct aacacatgca aacacatgca
agtcgagcga tgaagcttcc agtcgagcga tgaagcttccttcgggaagt ttcgggaagtggattagcgg ggattagcgg cggacgggtg cggacgggtg agtaacacgt agtaacacgt 120 gggtaacctg cctcaaagag gggtaacctg cctcaaagagtgggatagcc tgggatagcctcccgaaagg tcccgaaagg gagattaata gagattaata ccgcataaca ccgcataaca 180 180 ttattttatg gcatcataca ttattttatg gcatcatacataaaataatc taaaataatcaaaggagcaa aaaggagcaa tccgctttga tccgctttga gatggacccg gatggacccg 240 240 cggcgcatta gctagttggt cggcgcatta gctagttggtgaggtaacgg gaggtaacggctcaccaagg ctcaccaagg caacgatgcg caacgatgcg tagccgacct tagccgacct 300 300 gagagggtga tcggccacat gagagggtga tcggccacattggaactgag tggaactgagacacggtcca acacggtcca gactcctacg gactcctacg ggaggcagca ggaggcagca 360 360 gtggggaata ttncgcaatg gtggggaata ttncgcaatgggggaaaccc ggggaaaccctgacgcagca tgacgcagca acgccgcgtg acgccgcgtg agtgatgaag agtgatgaag 420 420 gttttcggat cgtaaaactc gttttcggat cgtaaaactctgtctttagg tgtctttagggacgataatg gacgataatg acggtaccta acggtaccta aggaggaagc aggaggaage 480 480 cacggctaac tacgtgccag cacggctaac tacgtgccagcagccgcggt cagccgcggtaatacgtagg aatacgtagg tggcaagcgt tggcaagcgt tgtccggatt tgtccggatt 540 540 tactgggcgt aaagagtatg tactgggcgt aaagagtatgtaggtgggtg taggtgggtgcttaagtcag cttaagtcag atgtgaaatt atgtgaaatt cccgggctca cccgggctca 600 600 acctgggagc tgcatttgaa acctgggage tgcatttgaaactgggcatc actgggcatctagagtgcag tagagtgcag gagaggaaag gagaggaaag tggaattcct tggaattect 660 660 agtgtagcgg tgaaatgcgt agtgtagcgg tgaaatgcgtagagattagg agagattaggaagaacacca aagaacacca gtggcgaagg gtggcgaagg cgactctctg cgactctctg 720 720 gactgtaact gacactgaga gactgtaact gacactgagatacgaaagcg tacgaaagcgtgggtagcaa tgggtagcaa acaggattag acaggattag ataccctggt ataccctggt 780 780 agtccacgnn gtaaacgatg agtccacgnn gtaaacgatgaatactaggt aatactaggtgtcggggggt gtcggggggt accaccctcg accaccctcg gtgccgcagc gtgccgcagc 840 840 aaacgcatta agtattccgc aaacgcatta agtattccgcctgggaagta ctgggaagtacggtcgcaag cggtcgcaag attaaaactc attaaaactc aaaggaattg aaaggaattg 900 900 acggggcccg cacaagcage acggggcccg cacaagcagcggagcatgtg ggagcatgtggtttaattcg gtttaattcg aagcaacgcg aagcaacgcg aagaacctta aagaacctta 960 960 cctagacttg acatctcctg cctagacttg acatctcctgaattactctt aattactcttaatcgaggaa aatcgaggaa gtcccttcgg gtcccttcgg ggacaggaag ggacaggaag 1020 acaggtggtg catggttgtc acaggtggtg catggttgtcgtcagctcgt gtcagctcgtgtcgtgagat gtcgtgagat gttgggttaa gttgggttaa gtcccgcaac gtcccgcaac 1080 1080 gagcgcaacc cttattgtta gagcgcaacc cttattgttagttgctacta gttgctactattaagttaag ttaagttaag cactctaacg cactctaacg agactgccgc agactgccgc 1140 1140 ggttaacgtg gaggaaggtg ggttaacgtg gaggaaggtggggatgacgt gggatgacgtcaaatcatca caaatcatca tgccccttat tgccccttat gtctagggct gtctagggct 1200 1200 acacacgtgc tacaatggct acacacgtgc tacaatggctggtacaacga ggtacaacgagcagcaaacc gcagcaaacc cgcgaggggg cgcgaggggg agcaaaactt agcaaaactt 1260 1260 gaaagccagt cccagttcgg gaaagccagt cccagttcggattgtaggct attgtaggctgaaactcgcc gaaactcgcc tacatgaagt tacatgaagt tggagttgct tggagttgct 1320 1320 agtaatcgcg aatcagcatg agtaatcgcg aatcagcatgtcgcggtgaa tcgcggtgaatacgttcccg tacgttcccg ggtcttgtac ggtcttgtac acaccgcccg acaccgcccg 1380 1380 tcacaccatg agagccggtaacacccgaag tcacaccatg agagccggta acacccgaagcccgtgaggt cccgtgaggt aaccgtaagg aaccgtaagg agccagcggt agccagcggt 1440 1440 cgaaggtggg attggtgatt cgaaggtggg attggtgattggggtaagtc ggggtaagtcgtaacaaggt gtaacaaggt agccgtagga agccgtagga gaacctgcgg gaacctgcgg 1500 1500 c t g g a t c a c c t c c t t t C t g g a t C a C C 1516 1516 tccttt <210> <210> 49 49 <211> <211> 1513 1513 <212> <212> DNA DNA <213> <213> Clostridium botulinum Clostridium botulinum
<220> <220> <221> misc_feature <221> misc_feature <222> <222> (1)..(7) (1)..(7) <223> n isa, <223> n is a,C,c,g, g,or ortt
<400> <400> 49 49 nnnnnnnaga gtttgatcct nnnnnnnaga gtttgatcctggctcaggac ggctcaggacgaacgctggc gaacgctggc ggcgtgccta ggcgtgccta acacatgcaa acacatgcaa
tcgagcgatg aagcttcctt cgggaagtgg tcgagcgatg aagcttcctt cgggaagtggattagcggcg attagcggcg gacgggtgag gacgggtgag taacacgtgg taacacgtgg 120 gtaacctgcc tcatagaggg gtaacctgcc tcatagaggggaatagectc gaatagcctcccgaaaggga ccgaaaggga gattaatacc gattaatacc gcataaagta gcataaagta 180 180 tgaaggtcgc atgacttcat tgaaggtcgc atgacttcattataccaaag tataccaaaggagtaatccg gagtaatccg ctatgagatg ctatgagatg gacccgcggc gacccgcggc 240 240 gcattagcta gttggtgagg gcattagcta gttggtgaggtaagggctca taagggctcaccaaggcaac ccaaggcaac gatgcgtagc gatgcgtagc cgacctgaga cgacctgaga 300 300 gggtgatcgg ccacattgga gggtgatcgg ccacattggaactgagacac actgagacacggtccagact ggtccagact cctacgggag cctacgggag gcagcagtgg gcagcagtgg 360 360 ggaatattgc gcaatggggg ggaatattgc gcaatgggggaaaccctgac aaaccctgacgcagcaacgc gcagcaacgc cgcgtgaatg cgcgtgaatg aagaaggcct aagaaggcct 420 420 tagggttgta aagttctgtc atatgggaag tagggttgta aagttctgtc atatgggaagataatgacgg ataatgacgg taccatatga taccatatga ggaagccacg ggaagccacg 480 480 gctaactacg tgccagcage gctaactacg tgccagcagccgcggtaata cgcggtaatacgtaggtggc cgtaggtggc aagcgttgtc aagcgttgtc cggatttact cggatttact 540 540 gggcgtaaag gatgcgtagg gggcgtaaag gatgcgtaggcggacattta cggacatttaagtcagatgt agtcagatgt gaaatacccg gaaatacccg ggctcaactt ggctcaactt 600 600 gggtgctgca tttgaaactg gggtgctgca tttgaaactgggtgtctaga ggtgtctagagtgcaggaga gtgcaggaga ggaaagcgga ggaaagcgga attcctagtg attcctagtg 660 660 tagcggtgaa atgcgtagag attaggaaga tagcggtgaa atgcgtagag attaggaagaacaccagtgg acaccagtgg cgaaggcggc cgaaggcggc tttctggact tttctggact 720 720 gtaactgacg ctgaggcatg gtaactgacg ctgaggcatgaaagcgtggg aaagcgtggggagcaaacag gagcaaacag gattagatac gattagatac cctggtagtc cctggtagtc 780 780 cacgccgtaa acgatgaatactaggtgtag cacgccgtaa acgatgaata ctaggtgtaggaggtatcga gaggtatcga ccccttctgt ccccttctgt gccgcagtta gccgcagtta 840 840 acacaataag tattccgcct acacaataag tattccgcctggggagtacg ggggagtacgatcgcaagat atcgcaagat taaaactcaa taaaactcaa aggaattgac aggaattgac 900 900 gggggcccgc acaagcagcg gggggcccgc acaagcagcggagcatgtgg gagcatgtggtttaattcga tttaattcga agcaacgcga agcaacgcga agaaccttac agaaccttac 960 960 ctagacttga catcccctga ctagacttga catcccctgaattacctgta attacctgtaatgagggaag atgagggaag cccttcgggg cccttcgggg cagggagaca cagggagaca 1020 ggtggtgcat ggttgtcgtc ggtggtgcat ggttgtcgtcagctcgtgtc agctcgtgtcgtgagatgtt gtgagatgtt gggttaagtc gggttaagtc ccgcaacgag ccgcaacgag 1080 1080 cgcaaccctt atcattagtt cgcaaccctt atcattagttgctaccatta gctaccattaagttgagcac agttgagcac tctagtgaga tctagtgaga ctgcccgggt ctgcccgggt 1140 1140 taaccgggag gaaggtgggg taaccgggag gaaggtggggatgacgtcaa atgacgtcaaatcatcatgc atcatcatgc cccttatgtc cccttatgtc tagggctaca tagggctaca 1200 1200 cacgtgctac aatggttggt cacgtgctac aatggttggtacaacaagat acaacaagatgcaagaccgc gcaagaccgc gaggtggagc gaggtggage taaacttaaa taaacttaaa 1260 1260 aaaccaacccagttcggatt aaaccaacc agttcggatt gtaggctgaa gtaggctgaa actcgcctac actcgcctac atgaagccgg atgaagccgg agttgctagt agttgctagt 1320 1320 aatcgcgaat cagcatgtcg aatcgcgaat cagcatgtcgcggtgaatac cggtgaatacgttcccgggc gttcccgggc cttgtacaca cttgtacaca ccgcccgtca ccgcccgtca 1380 1380 caccatgaga gctggtaaca caccatgaga gctggtaacacccgaagtcc cccgaagtccgtgaggtaac gtgaggtaac cgtaaggagc cgtaaggage cagcggccga cagcggccga 1440 1440 aggtgggatt agtgattggg aggtgggatt agtgattggggtgaagtcgt gtgaagtcgtaacaaggtag aacaaggtag ccgtaggaga ccgtaggaga acctgcggtt acctgcggtt 1500 1500 g g a t c a c c t c c t t g 1513g a t C a C C t C 1513 c t t
<210> <210> 50 50 <211> <211> 1521 1521 <212> <212> DNA DNA <213> <213> Francisellatularensis Francisella tularensis
<400> <400> 50 50 ttgaagagtt tgatcatggc tcagattgaa ttgaagagtt tgatcatggc tcagattgaacgctggtggc cgctggtggc atgcttaaca atgcttaaca catgcaagtc catgcaagte
gaacggtaac aggtcttagg gaacggtaac aggtcttaggatgctgacga atgctgacgagtggcggacg gtggcggacg ggtgagtaac ggtgagtaac gcgtaggaat gcgtaggaat 120 120
ctgcccattt gagggggata ctgcccattt gagggggataccagttggaa ccagttggaaacgactgtta acgactgtta ataccgcata ataccgcata atatctgtgg atatctgtgg 180 180
attaaaggtg gctttcggcg attaaaggtg gctttcggcgtgtcgcagat tgtcgcagatggatgageet ggatgagcct gcgttggatt gcgttggatt agctagttgg agctagttgg 240 tggggtaagg gcccaccaag tggggtaagg gcccaccaaggctacgatcc gctacgatccatagctgatt atagctgatt tgagaggatg tgagaggatg atcagccaca atcagccaca 300 300 ttgggactga gacacggcccaaactcctac ttgggactga gacacggccc aaactcctacgggaggcage gggaggcagc agtggggaat agtggggaat attggacaat attggacaat 360 360 gggggcaacc ctgatccago gggggcaacc ctgatccagcaatgccatgt aatgccatgtgtgtgaagaa gtgtgaagaa ggccctaggg ggccctaggg ttgtaaagca ttgtaaagca 420 420 ctttagttgg ggaggaaagcctcaaggtta ctttagttgg ggaggaaage ctcaaggttaatagccttgg atagccttgg gggaggacgt gggaggacgt tacccaaaga tacccaaaga 480 480 ataagcaccg gctaactccg ataagcaccg gctaactccgtgccagcage tgccagcagccgcggtaata cgcggtaata cggggggtgc cggggggtgc aagcgttaat aagcgttaat 540 540 cggaattact gggcgtaaag cggaattact gggcgtaaagggtctgtagg ggtctgtaggtggtttgtta tggtttgtta agtcagatgt agtcagatgt gaaagcccag gaaagcccag 600 600 ggctcaacct tggaactgca ggctcaacct tggaactgcatttgatactg tttgatactggcaaactaga gcaaactaga gtacggtaga gtacggtaga ggaatgggga ggaatgggga 660 660 atttctggtg tagcggtgaa atttctggtg tagcggtgaaatgcgtagag atgcgtagagatcagaagga atcagaagga acaccaatgg acaccaatgg cgaaggcaac cgaaggcaac 720 720 attctggacc gatactgaca attctggacc gatactgacactgagggacg ctgagggacgaaagcgtggg aaagcgtggg gatcaaacag gatcaaacag gattagatac gattagatac 780 780 cctggtagtc cacgctgtaa cctggtagtc cacgctgtaaacgatgagta acgatgagtactagctgttg ctagctgttg gagtcggtgt gagtcggtgt aaaggctcta aaaggctcta 840 840 gtggcgcacg taacgcgata gtggcgcacg taacgcgataagtactccgc agtactccgcctggggacta ctggggacta cggccgcaag cggccgcaag gctaaaactc gctaaaactc 900 900 aaaggaattg acggggaccc aaaggaattg acggggacccgcacaagcgg gcacaagcggtggagcatgt tggagcatgt ggtttaattc ggtttaattc gatgcaacgc gatgcaacgc 960 960 gaagaacctt acctggtctt gaagaacctt acctggtcttgacatcctgc gacatcctgcgaactttcta gaactttcta gagatagatt gagatagatt ggtgcttcgg ggtgcttcgg 1020 1020 aacgcagtga cagtgctgca aacgcagtga cagtgctgcacggctgtcgt cggctgtcgtcagctcgtgt cagctcgtgt tgtgaaatgt tgtgaaatgt tgggttaagt tgggttaagt 1080 1080 cccgcaacga gcgcaacccc cccgcaacga gcgcaacccctattgatagt tattgatagttaccatcatt taccatcatt aagttgggta aagttgggta ctctattaag ctctattaag 1140 actgccgctg acaaggcgga actgccgctg acaaggcggaggaaggtggg ggaaggtggggacgacgtca gacgacgtca agtcatcatg agtcatcatg gcccttacga gcccttacga 1200 1200 ccagggctac acacgtgcta caatgggtat ccagggctac acacgtgcta caatgggtattacagagggc tacagagggc tgcgaaggtg tgcgaaggtg cgagctggag cgagctggag 1260 1260 cgaaactcaa aaaggtacto cgaaactcaa aaaggtactcttagtccgga ttagtccggattgcagtctg ttgcagtctg caactcgact caactcgact gcatgaagtc gcatgaagtc 1320 1320 ggaatcgcta gtaatcgcag ggaatcgcta gtaatcgcaggtcagaatac gtcagaatactgcggtgaat tgcggtgaat acgttcccgg acgttcccgg gtcttgtaca gtcttgtaca 1380 1380 caccgcccgt cacaccatgg caccgcccgt cacaccatgggagtgggttg gagtgggttgctccagaagt ctccagaagt agatagctta agatagctta acgaatgggc acgaatgggc 1440 1440 gtttaccacg gagtgattca gtttaccacg gagtgattcatgactggggt tgactggggtgaagtcgtaa gaagtcgtaa caatggtagc caatggtage cgtagggaac cgtagggaac 1500 1500 c t g c g g c t g g a t c a c c t c c t t t ctgcggctgg 1521 1521 atcacctcct <210> <210> 51 51 <211> <211> 1464 1464 <212> <212> DNA DNA <213> <213> Vibrio cholerae Vibrio cholerae
<400> <400> 51 51 tggctcagat tgaacgctggcggcaggcct tggctcagat tgaacgctgg cggcaggcctaacacatgca aacacatgca agtcgagcgg agtcgagcgg cagcacagag cagcacagag
gaacttgttc cttgggtggc gaacttgttc cttgggtggcgagcggcgga gagcggcggacgggtgagta cgggtgagta atgcctggga atgcctggga aattgcccgg aattgcccgg 120 120
tagaggggga taaccattgg tagaggggga taaccattggaaacgatggc aaacgatggctaataccgca taataccgca taacctcgta taacctcgta agagcaaagc agagcaaage 180 180
aggggacctt cgggccttgc aggggacctt cgggccttgcgctaccggat gctaccggatatgcccaggt atgcccaggt gggattagct gggattagct agttggtgag agttggtgag 240 240
gtaagggctc accaaggcga gtaagggctc accaaggcgacgatccctag cgatccctagctggtctgag ctggtctgag aggatgatca aggatgatca gccacactgg gccacactgg 300 300
aactgagaca cggtccagac aactgagaca cggtccagactcctacggga tcctacgggaggcagcagtg ggcagcagtg gggaatattg gggaatattg cacaatgggc cacaatgggc 360 gcaagcctga tgcagccatg gcaagcctga tgcagccatgccgcgtgtat ccgcgtgtatgaagaaggcc gaagaaggcc ttcgggttgt ttcgggttgt aaagtacttt aaagtacttt 420 420 cagtagggag gaaggtggtt cagtagggag gaaggtggttaagctaatac aagctaataccttaatcatt cttaatcatt tgacgttacc tgacgttacc tacagaagaa tacagaagaa 480 480 gcaccggcta actccgtgcc gcaccggcta actccgtgccagcagccgcg agcagccgcggtaatacgga gtaatacgga gggtgcaagc gggtgcaage gttaatcgga gttaatcgga 540 540 attactgggc gtaaagcgca attactgggc gtaaagcgcatgcaggtggt tgcaggtggtttgttaagtc ttgttaagtc agatgtgaaa agatgtgaaa gccctgggct gccctgggct 600 600 caacctagga atcgcatttg caacctagga atcgcatttgaaactgacaa aaactgacaagctagagtac gctagagtac tgtagagggg tgtagagggg ggtagaattt ggtagaattt 660 660 caggtgtagc ggtgaaatgc caggtgtage ggtgaaatgcgtagagatct gtagagatctgaaggaatac gaaggaatac cggtggcgaa cggtggcgaa ggcggccccc ggcggccccc 720 720 tggacagata ctgacactca tggacagata ctgacactcagatgcgaaag gatgcgaaagcgtggggagc cgtggggagc aaacaggatt aaacaggatt agataccctg agataccctg 780 780 gtagtccacg ccgtaaacga gtagtccacg ccgtaaacgatgtctacttg tgtctacttggaggttgtga gaggttgtga cctagagtcg cctagagtcg tggctttcgg tggctttcgg 840 840 agctaacgcg ttaagtagac agctaacgcg ttaagtagaccgcctgggga cgcctggggagtacggtcgc gtacggtcgc aagattaaaa aagattaaaa ctcaaatgaa ctcaaatgaa 900 900 ttgacggggg cccgcacaag cggtggagca ttgacggggg cccgcacaag cggtggagcatgtggtttaa tgtggtttaa ttcgatgcaa ttcgatgcaa cgcgaagaac cgcgaagaac 960 960 cttacctact cttgacatcctcagaagaga cttacctact cttgacatcc tcagaagagactggagacag ctggagacag tcttgtgcct tcttgtgcct tcgggaactg tcgggaactg 1020 1020 agagacaggt gctgcatggc agagacaggt gctgcatggctgtcgtcagc tgtcgtcagctcgtgttgtg tcgtgttgtg aaatgttggg aaatgttggg ttaagtcccg ttaagtcccg 1080 1080 caacgagcgc aacccttatc caacgagegc aacccttatccttgtttgcc cttgtttgccagcacgtaat agcacgtaat ggtgggaact ggtgggaact ccagggagac ccagggagac 1140 1140 tgccggtgat aaaccggagg tgccggtgat aaaccggaggaaggtgggga aaggtggggacgacgtcaag cgacgtcaag tcatcatggc tcatcatggc ccttacgagt ccttacgagt 1200 1200 agggctacac acgtgctaca agggctacac acgtgctacaatggcgtata atggcgtatacagagggcag cagagggcag cgataccgcg cgataccgcg aggtggagcg aggtggagcg 1260 aatctcacaa agtacgtcgt aatctcacaa agtacgtcgtagtccggatt agtccggattggagtctgca ggagtctgca actcgactcc actcgactcc atgaagtcgg atgaagtcgg 1320 1320 aatcgctagt aatcgcaaat cagaatgttg aatcgctagt aatcgcaaat cagaatgttgcggtgaatac cggtgaatac gttcccgggc gttcccgggc cttgtacaca cttgtacaca 1380 1380 ccgcccgtca caccatggga gtgggctgca ccgcccgtca caccatggga gtgggctgcaaaagaagcag aaagaagcag gtagtttaac gtagtttaac cttcgggagg cttcgggagg 1440 1440 a c g c t t g c c a c t t t g g g t a c t t g g acgcttgcca 1464 1464 ctttgggtac ttgg
<210> <210> 52 52 <211> <211> 1469 1469 <212> <212> DNA DNA <213> <213> Yersinia pestis Yersinia pestis
<400> <400> 52 52 ctggcggcag gcctaacaca tgcaagtcga ctggcggcag gcctaacaca tgcaagtcgagcggcaccgg gcggcaccgg gaagtagttt gaagtagttt actactttgc actactttgc
cggcgagcgg cggacgggtg agtaatgtct cggcgagcgg cggacgggtg agtaatgtctggggatctgc ggggatctgc ctgatggagg ctgatggagg gggataacta gggataacta 120 120
ctggaaacgg tagctaataccgcatgacct ctggaaacgg tagctaatac cgcatgacctcgcaagagca cgcaagagca aagtggggga aagtggggga ccttagggcc ccttagggcc 180 180
tcacgccatc ggatgaaccc tcacgccatc ggatgaacccagatgggatt agatgggattagctagtagg agctagtagg tggggtaatg tggggtaatg gctcacctag gctcacctag 240 240
gcgacgatcc ctagctggtc gcgacgatcc ctagctggtctgagaggatg tgagaggatgaccagccaca accagccaca ctggaactga ctggaactga gacacggtcc gacacggtcc 300 300
agactcctac gggaggcage agactcctac gggaggcagcagtggggaat agtggggaatattgcacaat attgcacaat gggcgcaagc gggcgcaagc ctgatgcagc ctgatgcagc 360 360
catgccgcgt gtgtgaagaa catgccgcgt gtgtgaagaaggccttcggg ggccttcgggttgtaaagca ttgtaaagca ctttcagcga ctttcagcga ggaggaaggg ggaggaaggg 420 420
gttgagttta atacgctcaa gttgagttta atacgctcaatcattgacgt tcattgacgttactcgcaga tactcgcaga agaagcaccg agaagcaccg gctaactccg gctaactccg 480 480
tgccagcagc cgcggtaata tgccagcage cgcggtaatacggagggtgc cggagggtgcaagcgttaat aagcgttaat cggaattact cggaattact gggcgtaaag gggcgtaaag 540 cgcacgcagg cggtttgtta agtcagatgt cgcacgcagg cggtttgtta agtcagatgtgaaatccccg gaaatccccg cgcttaacgt cgcttaacgt gggaactgca gggaactgca 600 600 tttgaaactg gcaagctaga tttgaaactg gcaagctagagtcttgtaga gtcttgtagaggggggtaga ggggggtaga attccaggtg attccaggtg tagcggtgaa tagcggtgaa 660 660 atgcgtagag atctggagga atgcgtagag atctggaggaataccggtgg ataccggtggcgaaggcggc cgaaggcggc cccctggaca cccctggaca aagactgacg aagactgacg 720 720 ctcaggtgcg aaagcgtggg ctcaggtgcg aaagcgtggggagcaaacag gagcaaacaggattagatac gattagatac cctggtagtc cctggtagtc cacgctgtaa cacgctgtaa 780 780 acgatgtcga cttggaggtt acgatgtcga cttggaggttgtgcccttga gtgcccttgaggcgtggctt ggcgtggctt ccggagctaa ccggagctaa cgcgttaagt cgcgttaagt 840 840 cgaccgcctg gggagtacgg cgaccgcctg gggagtacggccgcaaggtt ccgcaaggttaaaactcaaa aaaactcaaa tgaattgacg tgaattgacg ggggcccgca ggggcccgca 900 900 caagcggtgg agcatgtggt caagcggtgg agcatgtggtttaattcgat ttaattcgatgcaacgcgaa gcaacgcgaa gaaccttacc gaaccttacc tactcttgac tactcttgac 960 960 atccacagaa tttggcagag atccacagaa tttggcagagatgctaaagt atgctaaagtgccttcggga gccttcggga actgtgagac actgtgagac aggtgctgca aggtgctgca 1020 1020 tggctgtcgt cagctcgtgt tggctgtcgt cagctcgtgttgtgaaatgt tgtgaaatgttgggttaagt tgggttaagt cccgcaacga cccgcaacga gcgcaaccct gcgcaaccct 1080 1080 tatcctttgt tgccagcacg taatggtggg tatcctttgt tgccagcacg taatggtgggaactcaaggg aactcaaggg agactgccgg agactgccgg tgacaaaccg tgacaaaccg 1140 1140 gaggaaggtg gggatgacgt gaggaaggtg gggatgacgtcaagtcatca caagtcatcatggcccttac tggcccttac gagtagggct gagtagggct acacacgtgc acacacgtgc 1200 1200 tacaatggca gatacaaagt gaagcgaact tacaatggca gatacaaagt gaagcgaactcgcgagagcc cgcgagagcc agcggaccac agcggaccac ataaagtctg ataaagtctg 1260 1260 tcgtagtccg gattggagtc tcgtagtccg gattggagtctgcaactcga tgcaactcgactccatgaag ctccatgaag tcggaatcgc tcggaatcgc tagtaatcgt tagtaatcgt 1320 1320 agatcagaat gctacggtga agatcagaat gctacggtgaatacgttccc atacgttcccgggccttgta gggccttgta cacaccgccc cacaccgccc gtcacaccat gtcacaccat 1380 1380 gggagtgggt tgcaaaagaa gggagtgggt tgcaaaagaagtaggtagct gtaggtagcttaaccttcgg taaccttcgg gagggcgctt gagggcgctt accactttgt accactttgt 1440 gattcatgac tggggtgaag tcgtaacaa gattcatgac 1469 1469 tggggtgaag tcgtaacaa
<210> <210> 53 53 <211> <211> 24 24 <212> <212> DNA DNA <213> <213> Artificial Sequence Artificial Sequence
<220> <220> <223> Forward Primer <223> Forward Primer
<400> <400> 53 53 c a t c c a a g g a a g g c a g c a g g c g cc gg c g catccaagga 24 24 aggcagcagg <210> <210> 54 54 <211> <211> 22 22 <212> <212> DNA DNA <213> <213> ArtificialSequence Artificial Sequence
<220> <220> <223> Reverse Primer <223> Reverse Primer
<400> <400> 54 54 g t t c a a c t a c g a g c t t t t t a a c a C gttcaactac 22 22 gagcttttta <210> <210> 55 55 <211> <211> 22 22 <212> <212> DNA DNA <213> <213> Artificial Sequence Artificial Sequence
<220> <220> <223> Reverse Primer <223> Reverse Primer
<400> <400> 55 55 g t t c g a c t a c g a g c t t t t t a a c a C gttcgactac 22 22 gagcttttta <210> <210> 56
<211> <211> 23 23 <212> <212> DNA DNA <213> <213> Artificial Sequence Artificial Sequence
<220> <220> <223> ForwardPrimer <223> Forward Primer
<400> 56 <400> 56 g t g t a g c g g t g a a a t g c g t a g a g gtgtagcggt 23 23 gaaatgcgta gag
<210> <210> 57 57 <211> <211> 23 23 <212> <212> DNA DNA <213> <213> ArtificialSequence Artificial Sequence
<220> <220> <223> ReversePrimer <223> Reverse Primer
<400> 57 <400> 57 t c g t t t a c c g t g g a c t a c c a g g gg g g tcgtttaccg 23 23 tggactacca

Claims (1)

1. A method of identifying, partially identifying or classifying at least one bacterium in a sample, said method comprising analysing at least a portion of a bacterial 16S rRNA gene or gene product from the sample for the presence or absence of at least two single nucleotide polymorphisms (SNPs); wherein the at least two SNPs in the at least a portion of the bacterial 16S rRNA gene or gene product are at positions corresponding to at least two of positions 273, 378, 412, 440, 488, 647, 653, 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1; and wherein the at least one bacterium in the sample is identified, partially identified or classified based on the presence or absence of the at least two SNPs.
2. A method of identifying, partially identifying, classifying or diagnosing a bacterial infection in a subject, said method comprising analysing for the presence or absence of at least two single nucleotide polymorphisms (SNPs) in at least a portion of a bacterial 16S rRNA gene or gene product in a sample from the subject; wherein the at least two SNPs in the at least a portion of the bacterial 16S rRNA gene or gene product are at positions corresponding to at least two of positions 273, 378, 412, 440, 488, 647, 653, 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1; and wherein the presence or absence of said at least two SNPs in the at least a portion of the 16S rRNA gene or gene product is used to identify, partially identify, classify or diagnose the bacterial infection in the subject.
3. A method of treating a subject having a bacterial infection, said method comprising: identifying, partially identifying, classifying or diagnosing a bacterial infection in a subject according to the method of claim 2; and administering to the subject a therapy or treatment agent for treating the bacterial infection in the subject. 4. The method of any one of claims 1 to 3, wherein the at least two SNPs in the at least a portion of the bacterial 16S rRNA gene or gene product are at positions corresponding to at least two of positions 273, 378, 412, 440, 488, 647, and 653 of the 16S rRNA gene set forth in SEQ ID NO: 1. 5. The method of claim 4, wherein the bacterium is or the bacterial infection is caused by at least one organism selected from the group consisting of: Acinetobacter calcoaceticus; Enterobacter aerogenes; Enterobacter cloacae; Enterococcusfaecalis; Enterococcusfaecium; Escherichia coli; Klebsiella pneumoniae; Proteus mirabilis;Pseudomonas aeruginosa;Serratia marcescens; Staphylococcus aureus; Streptococcus agalactiae; Streptococcus pneumoniae; Streptococcuspyogenes; and Staphylococcus epidermidis. 6. A method of identifying, partially identifying or classifying at least one bacterium in a sample, said method comprising analysing at least a portion of a bacterial 16S rRNA gene or gene product from the sample for the presence or absence of at least one single nucleotide polymorphism (SNP); wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is at a position corresponding to at least one of positions 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1; and wherein the at least one bacterium in the sample is identified, partially identified or classified based on the presence or absence of the at least one SNP. 7. A method of identifying, partially identifying, classifying or diagnosing a bacterial infection in a subject, said method comprising analysing for the presence or absence of at least one single nucleotide polymorphism (SNP) in at least a portion of a bacterial 16S rRNA gene or gene product in a sample from the subject; wherein the at least one SNP in the at least a portion of the bacterial 16S rRNA gene or gene product is at a position corresponding to at least one of positions 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1; and wherein the presence or absence of said at least one SNP in the at least a portion of the 16S rRNA gene or gene product is used to identify, partially identify, classify or diagnose the bacterial infection in the subject. 8. The method of claim 6 or claim 7, wherein the bacterium is or the bacterial infection is caused by at least one organism selected from the group consisting of: Bacillus anthracis, Clostridium botulinum, Yersinia pestis, Francisella tularensis, Vibrio cholerae, and Burkholderiapseudomallei. 9. The method of any one of claims 1 to 8, wherein the sample is a biological sample comprising sputum, blood, cerebrospinal fluid or urine. 10. The method of any one of claims 1 to 9, wherein the step of analysing comprises determining the presence or the absence of the SNP or SNPs using high resolution melt analysis, 5' nuclease digestion, molecular beacons, oligonucleotide ligation, microarray, restriction fragment length polymorphism, antibody detection methods, direct sequencing or any combination thereof. 11. The method of any one of claims 1 to 10, wherein said method further comprises the step of determining whether the at least one bacterium is resistant to a therapeutic agent.
12. A method of identifying, partially identifying, or classifying at least one bacterium in a sample, said method comprising: combining with the sample at least one oligonucleotide comprising a nucleotide sequence as set forth in at least one of SEQ ID NOs: 22, 25-28, 33-35, and 57, or at least one oligonucleotide consisting of a nucleotide sequence as set forth in at least one of SEQ ID NOs: 16-35, 56 and 57; and identifying, partially identifying, or classifying the at least one bacterium based on the presence or absence of at least two single nucleotide polymorphisms (SNPs) in at least a portion of a bacterial 16S rRNA gene or gene product, wherein the at least two SNPs in the at least a portion of the bacterial 16S rRNA gene or gene product are at positions corresponding to at least two of positions 273, 378, 412, 440, 488, 647, 653, 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1. 13. An array comprising more than one oligonucleotide comprising a nucleotide sequence as set forth in at least one of SEQ ID NOs: 22, 25-28, 33-35, and 57, or more than one oligonucleotide consisting of a nucleotide sequence as set forth in at least one of SEQ ID NOs: 16-35, 56 and 57. 14. A biochip comprising a solid substrate and at least one oligonucleotide comprising a nucleotide sequence as set forth in at least one of SEQ ID NOs: 22, 25-28, 33-35, and 57, or at least one oligonucleotide consisting of a nucleotide sequence as set forth in at least one of SEQ ID NOs: 16-35, 56 and 57. 15. A kit or assay when used according to the method of any one of claims I to 12, said kit or assay comprising: an oligonucleotide comprising a nucleotide sequence as set forth in at least one of SEQ ID NOs: 22, 25-28, 33-35, and 57, or an oligonucleotide consisting of a nucleotide sequence as set forth in at least one of SEQ ID NOs: 16-35, 56 and 57; the array of claim 13; and/or the biochip of claim 14. 16. The method of any one of claims 1 to 3 and 12, wherein the at least two SNPs are at least four SNPs at positions corresponding to at least four of positions 273, 378, 412, 440, 488, 647, 653, 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1. 17. The method of any one of claims 1 to 3 and 12, wherein the at least two SNPs are at least five SNPs at positions corresponding to at least five of positions 273, 378, 412, 440, 488, 647, 653, 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1. 18. The method of any one of claims I to 5 and 12, wherein the at least two SNPs are seven SNPs at positions corresponding to positions 273, 378, 412, 440, 488, 647 and 653 of the 16S rRNA gene set forth in SEQ ID NO: 1.
19. The method of any one of claims 6 to 8, wherein the at least one SNP is four SNPs at positions corresponding to positions 737, 755, 762 and 776 of the 16S rRNA gene set forth in SEQ ID NO: 1.
aureus Staphylococcus TTTTATGGAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCA epidermidis Staphylococcus PTTATGGAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGC) pneumoniae Streptococcus ----GTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCA WO
agalactiae Streptococcus ---GACGAACGCTGGCGGCGTGCCTAATACATGCA --GAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCA pyogenes Streptococcus faecalis Enterococcus AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCA faecium Enterococcus PAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCT-ATACATG mirabilis Proteus TGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCI -AAATTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCA coli Escherichia marcescens Serratia GCTCAGATTGAACGCTGGCGGCAGGCTTAACACATGCE aerogenes Enterobacter -ACGCTGGCGGCAGGCCTAACACATGCA cloacae Enterobacter TGAACGCTGGCGGCAGGCCTAACACATGCA pneumoniae Klebsiella -AGAGTTTGATNNTGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCA aeruginosa Pseudomonas TGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGO calcoaceticus Acinetobacter TAGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCTTAACACATGCA *
*
aureus Staphylococcus CTTCTCTGATGTTAGCGGCGGACG AGTCGAGCGAACGGACGAGAAGCTTG epidermidis Staphylococcus -CTTCTCTGACGTTAGCGGCGGACG AGTCGAGCGAACAGACGAGGAGCTTG Substitute pneumoniae Streptococcus AGTAGAACGCTGAAGGAGGAGCTT GCTTCTCTGGATGAGTTGCGAACG agalactiae Streptococcus AGTAGAACGCTGAGGTTTGGTGTTT ACACTAGACTGATGAGTTGCGAACG Sheet GCACCGGTTCAAGGAGTTGCGAACG AGTAGAACGCTGAGAACTGGTGCTT pyogenes Streptococcus faecalis Enterococcus AGTCGAACGCTTCTTTCCTCCCGAGTGCTTGCACTCAATTGGAAAGAGGAGTGGCGGACG AGTCGAACGCTTCTTTTTCCACCGGAGCTTGCTCCACCGGAAAAAGAGGAGTGGCGAACG faecium Enterococcus mirabilis Proteus AGTCGAGCGGTAACAGGAGAAAGCTTGCTTTCTTGC TGACGAGCGGCGGACG coli Escherichia AGTCGAACGGTAACAGGAAGAAGCTTGCTTCTTTGC TGACGAGTGGCGGACG AGTCGAGCGGTAGCACAGGGGAGCTTGCTCCCTGGG marcescens Serratia TGACGAGCGGCGGACG AGTCGAGCGGTAGCACAGAGAGCT--TGCTCTCGG aerogenes Enterobacter TGACGAGCGGCGGACG cloacae Enterobacter AGTCGAACGGTAGCACAGAGAGCT--TGCTCTCGGG TGACGAGTGGCGGACG pneumoniae Klebsiella AGTCGAGCGGTAGCACAGAGAGCT--TGCTCTCGGG TGACGAGCGGCGGACG aeruginosa Pseudomonas GATTCAGCGGCGGACG AGTCGAGCGGATGAAGGGAGCTTGCT CCTG
calcoaceticus Acinetobacter ACTG AGTCGAGCGGGGAAAGGTAGCTTGCT GACCTAGCGGCGGACG *** *********
Figure 1 aureus Staphylococcus GGTGAGTAACACGTGGATAACCTACCTATAAGACTGGGATAACTTCGGGAAACCGGAG0 epidermidis Staphylococcus GGTGAGTAACACGTGGATAACCTACCTATAAGACTGGGATAACTTCGGGAAACCGGAGC GGTGAGTAACGCGTAGGTAACCTGCCTGGTAGCGGGGGATAACTATTGGAAACGATAGC pneumoniae Streptococcus WO agalactiae Streptococcus GGTGAGTAACGCGTAGGTAACCTGCCTCATAGCGGGGGATAACTATTGGAAACGATAGCT GGTGAGTAACGCGTAGGTAACCTACCTCATAGCGGGGGATAACTATTGGAAACGATAGO pyogenes Streptococcus GGTGAGTAACACGTGGGTAACCTACCCATCAGAGGGGGATAACACTTGGAAACAGGTGCT faecalis Enterococcus GGTGAGTAACACGTGGGTAACCTGCCCATCAGAAAGGGATAACACTTGGAAACAGGTGCT faecium Enterococcus mirabilis Proteus GGTGAGTAATGTATG-GGGATCTGCCCGATAGAGGGGGATAACTACTGGAAACGGTGGCT ORDERS
GGTGAGTAATGTCTG-GGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCT coli Escherichia GGTGAGTAATGTCTG-GGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCT marcescens Serratia GGTGAGTAATGTCTG-GGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCT aerogenes Enterobacter cloacae Enterobacter cloacae Enterobacter GGTGAGTAATGTCTG-GGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGC pneumoniae Klebsiella GGTGAGTAATGTCTG-GGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCT aeruginosa Pseudomonas GGTGAGTAATGCCTA-GGAATCTGCCTGGTAGTGGGGGATAACGTCCGGAAACGGGCGCT calcoaceticus Acinetobacter GGTGAGTAATGCTTA-GGAATCTGCCTATTAGTGGGGGACAACATTCCGAAAGGAATGCT *** ***
* ****
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* AAGACGGTCTTGCTGTCA AATACCGGATAATATTTTGAACCGCATGGTTCAAAAGTGA- aureus Staphylococcus epidermidis Staphylococcus AAGACGGTTTTGCTGTCA AATACCGGATAATATATTGAACCGCATGGTTCAATAGTGA- AATACCGCATAAGAGTGGATGTTGCATGACAT-TTGCTTAAAA--GGTGCACTTGCATCA pneumoniae Streptococcus Substitute Sheet agalactiae Streptococcus -GGAGCAATTGCTTCA AATACCGCATAAGAGTAATTAACACATGTTAG-TTATTTAAAA-- AATACCGCATAAGAGAGACTAACGCATGTTAG-TAATTTAAAA--GGGGCAATTGCTCCA pyogenes Streptococcus AATACCGCATAACAGTTTAT-GCCGCATGGCATAAGAGTGAAAGGCGCTTTCGGGTGTCG faecalis Enterococcus AATACCGTATAACAAATCAAAACCGCATGGTTTTGATTTGAAAGGCGCTTTCGGGTGTC faecium Enterococcus mirabilis Proteus AGGGGCTCTTCGGACCTTGCA AATACCGCATAATGTCTACGGACCAAAG coli Escherichia AATACCGCATAACGTCGCAAGACCAAAG PAGGGGGACCTTCGGGCCTCTTG marcescens Serratia AATACCGCATAACGTCGCAAGACCAAAG AGGGGGACCTTCGGGCCTCTTG aerogenes Enterobacter AATACCGCATAACGTCGCAAGACCAAAG STGGGGGACCTTCGGGCCTCATG cloacae Enterobacter AATACCGCATAAYGTCGCAAGACCAAAG AGGGGGACCTTCGGGCCTCTTG pneumoniae Klebsiella AATACCGCATAACGTCGCAAGACCAAAG TGGGGGACCTTCGGGCCTCATG aeruginosa Pseudomonas AATACCGCATACGTCCTGAGGGAGAAAG TGGGGGATCTTCGGACCTCACG calcoaceticus Acinetobacter AATACCGCATACGTCCTACGGGAGAAAG CAGGGGACCTTCGGGCCTTGCG (continued) 1 Figure
SNP273 CTTATAGATGGATCCGCGCTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCA aureus Staphylococcus epidermidis Staphylococcus CTTATAGATGGATCCGCGCCGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAL CTACCAGATGGACCTGCGTTGTATTAGCTAGTTGGTGGGGTAACGGCTCACCAAGGCGA0 pneumoniae Streptococcus WO
agalactiae Streptococcus CTGTGAGATGGACCTGCGTTGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGA pyogenes Streptococcus TATGAGATGGACCTGCGTTGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGZ TTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCA0 faecalis Enterococcus faecium Enterococcus CTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCA0 mirabilis Proteus CTATCGGATGAACCCATATGGGATTAGCTAGTAGGTGGGGTAAAGGCTCACCTAGGCGA0 CCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGA coli Escherichia CCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAATGGCTCACCT/ marcescens Serratia CCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAATGGCTCACCE aerogenes Enterobacter CCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGA cloacae Enterobacter pneumoniae Klebsiella CCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTA aeruginosa Pseudomonas CTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAG calcoaceticus Acinetobacter CTAATAGATGAGCCTAAGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGAC *****
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aureus Staphylococcus GATGCATAGCCGACC-TGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAGAG epidermidis Staphylococcus GATGCGTAGCCGACC-TGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAGAC GATACATAGCCGACC-TGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGA0 pneumoniae Streptococcus Submitters: GATACATAGCCGACC-TGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGAC agalactiae Streptococcus ATACATAGCCGACC-TGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGAO pyogenes Streptococcus ATGCATAGCCGACC-TGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGAC faecalis Enterococcus faecium Enterococcus GATGCATAGCCGCACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCA-AL mirabilis Proteus ATCTCTAGCTGGTC-TGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGA GATCCCTAGCTGGTC-TGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGA0 coli Escherichia GATCCCTAGCTGGTC-TGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGAG marcescens Serratia ATCCCTAGCTGGTC-TGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGAC aerogenes Enterobacter cloacae Enterobacter GATCCCTAGCTGGTC-TGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGAC pneumoniae Klebsiella GATCCCTAGCTGGTC-TGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGA0 aeruginosa Pseudomonas ATCCGTAACTGGTC-TGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGAC GATCTGTAG-CGGTC-TGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGAC calcoaceticus Acinetobacter ***
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* *
(continued) 1 Figure
SNP378 TCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAAC aureus Staphylococcus epidermidis Staphylococcus CCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACO TCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGGAAGTCTGACCGAGCAA pneumoniae Streptococcus WO
PCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGGAAGTCTGACCGAGCAAC agalactiae Streptococcus TCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACG pyogenes Streptococcus TCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACG faecalis Enterococcus CTCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACG faecium Enterococcus TCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATG mirabilis Proteus TCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATG coli Escherichia CCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCA marcescens Serratia CCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCZ aerogenes Enterobacter TCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATG cloacae Enterobacter TCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATG pneumoniae Klebsiella TCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATO aeruginosa Pseudomonas TCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGGAACCCTGATCCAGCCATG calcoaceticus Acinetobacter * * *** **** ** * ****** ** ****** SNP412 SNP440
CCGCGTGAGTGATGAAGGTCTTCGGATCGTAAAACTCTGTTATTAGGGAAGAACATATGT aureus Staphylococcus 4126
26) CCGCGTGAGTGATGAAGGTCTTCGGATCGTAAAACTCTGTTATTAGGGAAGAACAAATG epidermidis Staphylococcus CCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTAAGAGAAGAACGAGTGT pneumoniae Streptococcus CGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGAGAAGAACGTTGGT Substitute Sheet agalactiae Streptococcus Description CCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGAGAAGAATGATGGT pyogenes Streptococcus CCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGAC. faecalis Enterococcus CCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGAT faecium Enterococcus SCGCGTGTATGAAGAAGGCCTTAGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGTGATA. mirabilis Proteus GTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTA coli Escherichia SCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAGGAAGGTG marcescens Serratia CCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGAGGAGGAAGGCGTT aerogenes Enterobacter CCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGTGT cloacae Enterobacter CCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGATG pneumoniae Klebsiella CCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGT aeruginosa Pseudomonas CCGCGTGTGTGAAGAAGGCCTTATGGTTGTAAAGCACTTTAAGCGAGGAGGAGGCTACTA calcoaceticus Acinetobacter ** * ** ***** ***** ******* (continued) 1 Figure
SNP488 GTAAGTAAC-TGTGCACATCTTGACGGTACCTAATCAGAAAGCCACGGCTAACTACGTGO aureus Staphylococcus epidermidis Staphylococcus AAGTAAC-TATGCACGTCTTGACGGTACCTAATCAGAAAGCCACGGCTAACTACGTO GAGAGTGGAAAGTTCACACTGTGACGGTATCTTACCAGAAAGGGACGGCTAACTACGTGC pneumoniae Streptococcus WO
agalactiae Streptococcus AGGAGTGGAAAATCTACCAAGTGACGGTAACTAACCAGAAAGGGACGGCTAACTACGTGO GGGAGTGGAAAATCCACCAAGTGACGGTAACTAACCAGAAAGGGACGGCTAACTACGTGC pyogenes Streptococcus TTAGTAAC-TGAACGTCCCCTGACGGTATCTAACCAGAAAGCCACGGCTAACTACGTGO faecalis Enterococcus GAGAGTAAC-TGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTACGTG0 faecium Enterococcus AGGTTAATACCCTTGTCAATTGACGTTACC-CGCAGAAGAAGCACCGGCTAACTCCGTGC mirabilis Proteus AGTTAATACCTTTGCTCATTGACGTTACC-CGCAGAAGAAGCACCGGCTAACTCCGTGC coli Escherichia AGCTTAATACGTTCATCAATTGACGTTACT-CGCAGAAGAAGCACCGGCTAACTCCGTGC marcescens Serratia AGGTTAATAACCTTGGCGATTGACGTTACT-CGCAGAAGAAGCACCGGCTAACTCCGTGC aerogenes Enterobacter TGGTTAATAACCGCAGCAATTGACGTTACC-CGCAGAAGAAGCACCGGCTAACTCCGTGC cloacae Enterobacter AGGTTAATAACCTCATCGATTGACGTTACCCTGCAGAAGAAGCACCGGCTAACTCCGTGC pneumoniae Klebsiella AAGTTAATAC-CTTGCTGTTTTGACGTTACCAACAGAATAAGCACCGGCTAACTTCGTGC aeruginosa Pseudomonas GTATTAATACTACTGGATAGTGGACGTTACTCGCAGAATAAGCACCGGCTAACTCTGTGC calcoaceticus Acinetobacter * ****
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aureus Staphylococcus CAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGO epidermidis Staphylococcus CAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCG0 CAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGA pneumoniae Streptococcus Subtotal: CAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGA agalactiae Streptococcus CAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCG ROIAL pyogenes Streptococcus faecalis Enterococcus CAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGA CAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCG faecium Enterococcus CAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGO mirabilis Proteus CAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCO coli Escherichia CAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGC marcescens Serratia CAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCG0 aerogenes Enterobacter cloacae Enterobacter CAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGO CAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGC pneumoniae Klebsiella AGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGO aeruginosa Pseudomonas CAGCAGCCGCGGTAATACAGAGGGTGCGAGCGTTAATCGGATTTACTGGGCGTAAAGCG? calcoaceticus Acinetobacter * Figure 1 (continued) INSURANCE
GCGTAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTG aureus Staphylococcus GCGTAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATT epidermidis Staphylococcus GCGCAGGCGGTTAGATAAGTCTGAAGTTAAAGGCTGTGGCTTAACCATAGT-AGGCTTTO pneumoniae Streptococcus WO
GCGCAGGCGGTTCTTTAAGTCTGAAGTTAAAGGCAGTGGCTTAACCATTGT-ACGCTTTG agalactiae Streptococcus GCGCAGGCGGTTTTTTAAGTCTGAAGTTAAAGGCATTGGCTCAACCAATGT-ACGCTTT pyogenes Streptococcus GCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTO faecalis Enterococcus GCAGGCGG-TTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATT faecium Enterococcus ACGCAGGCGGTCAATTAAGTCAGATGTGAAAGCCCCGAGCTTAACTTGGGAATTGCATC mirabilis Proteus ACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCT coli Escherichia ACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATT marcescens Serratia ACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATT aerogenes Enterobacter ACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATT cloacae Enterobacter ACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTO pneumoniae Klebsiella GCGTAGGTGGTTCAGCAAGTTGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATO aeruginosa Pseudomonas GCGTAGGCGGCCATTTAAGTCAAATGTGAAATCCCCGAGCTTAACTTGGGAATTGCATTG calcoaceticus Acinetobacter **** *** ***
* **
*
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SNP647 SNP653 GAAACTGGAAAACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATO aureus Staphylococcus epidermidis Staphylococcus AAACTGGAAAACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATG GAAACTGTTTAACTTGAGTGCAAGAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATG pneumoniae Streptococcus Submitter: Sheet GAAACTGGAGGACTTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATG agalactiae Streptococcus GAAACTGGAGAACTTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATG pyogenes Streptococcus GAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATG faecalis Enterococcus GAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAAT faecium Enterococcus GAAACTGGTTGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCATGTGTAGCGGTGAAATO mirabilis Proteus GATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATG coli Escherichia GAAACTGGCAAGCTAGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATG marcescens Serratia GAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATG aerogenes Enterobacter GAAACTGGCAGGCTGGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATG cloacae Enterobacter pneumoniae Klebsiella GAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATG AAAACTACTGAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATG aeruginosa Pseudomonas CATACTGGATGGCTAGAGTATGGGAGAGGATGGTAGAATTCCAGGTGTAGCGGTGAAATO calcoaceticus Acinetobacter * *** * MEMBERSHIP
(continued) 1 Figure
CGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACGCTG aureus Staphylococcus epidermidis Staphylococcus CGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACGCT CGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGCTTGTAACTGACGCT pneumoniae Streptococcus WO
agalactiae Streptococcus CGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCT CGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTG pyogenes Streptococcus CGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTO faecalis Enterococcus CGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTG faecium Enterococcus CGTAGAGATGTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTC mirabilis Proteus CGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCT COLI ESCHERICHIA CGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGGCCCCTGGACGAAGACTGACGCTO marcescens Serratia aerogenes Enterobacter CGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCT< CGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCT cloacae Enterobacter pneumoniae Klebsiella CGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCT CGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACT aeruginosa Pseudomonas CGTAGAGATCTGGAGGAATACCGATGGCGAAGGCAGCCATCTGGCCTAATACTGACGCTG calcoaceticus Acinetobacter ***** *** *******
* ****
** aureus Staphylococcus ATGTGCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAAG epidermidis Staphylococcus ATGTGCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACG AGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAAC pneumoniae Streptococcus agalactiae Streptococcus AGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACO AGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAAC pyogenes Streptococcus faecalis Enterococcus AGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACO faecium Enterococcus AGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAAC AGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACO mirabilis Proteus AGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAAG coli Escherichia AGGTGCCAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACG marcescens Serratia AGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAAG aerogenes Enterobacter cloacae Enterobacter AGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACO pneumoniae Klebsiella AGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACG AGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAAG aeruginosa Pseudomonas AGGTACGAAAGCATGGGAGCAGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACG calcoaceticus Acinetobacter **********
(continued) 1 Figure aureus Staphylococcus ATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCA0 epidermidis Staphylococcus ATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCAO ATGAGTGCTAGGTGTTAGACCCTTTCCGGGGTTTAGTGCCGTAGCTAACGCATTAAGCAO pneumoniae Streptococcus WV agalactiae Streptococcus ATGAGTGCTAGGTGTTAGGCCCTTTCCGGGGCTTAGTGCCGCAGCTAACGCATTAAGCA0 pyogenes Streptococcus ATGAGTGCTAGGTGTTAGGCCCTTTCCGGGGCTTAGTGCCGGAGCTAACGCATTAAGCAO faecalis Enterococcus ATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCAAACGCATTAAGCA0 faecium Enterococcus ATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCA mirabilis Proteus ATGTCGATTTAGAGGTTGTGGTCTTG-AACCGTGGCTTCTGGAGCTAACGCGTTAAATC coli Escherichia ATGTCGACTTGGAGGTTGTGCCCTTG-AGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCG marcescens Serratia ATGTCGATTTGGAGGTTGTGCCCTTG-AGGCGTGGCTTCCGGAGCTAACGCGTTAAATC aerogenes Enterobacter ATGTCGACTTGGAGGTTGTGCCCTTG-AGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCG cloacae Enterobacter ATGTCGATTTGGAGGTTGTGCCCTTG-AGGCGTGGCTTCCGGAGCTAACGCGTTAAATC pneumoniae Klebsiella ATGTCGATTTGGAGGTTGTGCCCTTG-AGGCGTGGCTTCCGGAGCTAACGCGTTAAATC aeruginosa Pseudomonas ATGTCGACTAGCCGTTGGGATCCTTG-AGATCTTAGTGGCGCAGCTAACGCGATAAGTCG calcoaceticus Acinetobacter ATGTTTACTAGCCGTTGGGGCCTTTG-AGGCTTTAGTGGCGCAGCTAACGCGATAAGTAG *****
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* * aureus Staphylococcus TCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCAC epidermidis Staphylococcus TCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACA TCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCAC pneumoniae Streptococcus agalactiae Streptococcus TCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACA pyogenes Streptococcus TCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACA faecalis Enterococcus TCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCAC faecium Enterococcus TCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACA mirabilis Proteus ACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACA coli Escherichia ACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACA marcescens Serratia ACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCAC ACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCAC aerogenes Enterobacter cloacae Enterobacter ACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACA pneumoniae Klebsiella CCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCAC aeruginosa Pseudomonas ACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACA calcoaceticus Acinetobacter ACCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAATGAATTGACGGGGGCCCGCACA *
* Figure 1 (continued)
GCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAATCTTGACZ aureus Staphylococcus AGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAATCTTGAC epidermidis Staphylococcus AGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACAT pneumoniae Streptococcus AGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACZ agalactiae Streptococcus AGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACA pyogenes Streptococcus AGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACAT faecalis Enterococcus AGCGGTGGAGCATGTGGTTTAATTCGAAGCAACACGAAGAACCTTACCAGGTCTTGAC faecium Enterococcus WO03/05/2020
AGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGAC. mirabilis Proteus AGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACAT coli Escherichia GCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACE marcescens Serratia AGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACE aerogenes Enterobacter GCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACA cloacae Enterobacter AGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACAT pneumoniae Klebsiella GCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTGGCCTTGACZ aeruginosa Pseudomonas AGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGCCTTGACAT calcoaceticus Acinetobacter ***** CCTTTGACAACTCTAGAGATAGAGCTTTCCCCTTCGGGGGACAAAGTGACAGGTGGTGC/ aureus Staphylococcus CCTCTGACCCCTCTAGAGATAGAGTTTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGC epidermidis Staphylococcus CCCTCTGACCGCTCTAGAGATAGAGTTTT--CCTTCGGGACAGAGGTGACAGGTGGTGC) Subscriptions pneumoniae Streptococcus CCTTCTGACCGGCCTAGAGATAGGCTTTC--TCTTCGGAGCAGAAGTGACAGGTGGTGCA agalactiae Streptococcus Sheet CCCGATGCCCGCTCTAGAGATAGAGTTTT--ACTTCGGTACATCGGTGACAGGTGGTGCA pyogenes Streptococcus CCTTTGACCACTCTAGAGATAGAGCTTTC--CCTTCGGGGACAAAGTGACAGGTGGTGC/ faecalis Enterococcus CCTTTGACCACTCTAGAGATAGAGCTTCC--CCTTCGGGGGCAAAGTGACAGGTGGTGCA faecium Enterococcus CCAGCGAATCCTTTAGAGATAGAGGAGTG--CCTTCGGGAACGCTGAGACAGGTGCTGCA mirabilis Proteus CCACAGAACTTTCCAGAGATGGATTGGTG--CCTTCGGGAACTGTGAGACAGGTGCTGCA coli Escherichia CCAGAGAACTTTCCAGAGATGGATTGGTG--CCTTCGGGAACTCTGAGACAGGTGCTGCA marcescens Serratia CCAGAGAACTTAGCAGAGATGCTTTGGTG--CCTTCGGGAACTCTGAGACAGGTGCTGCA aerogenes Enterobacter CCACAGAACTTTCCAGAGATGGATTGGTG--CCTTCGGGAACTGTGAGACAGGTGCTGCA cloacae Enterobacter CCACAGAACTTTCCAGAGATGGATTGGTG--CCTTCGGGAACTGTGAGACAGGTGCTGC. pneumoniae Klebsiella GCTGAGAACTTTCCAGAGATGGATTGGTG--CCTTCGGGAACTCAGACACAGGTGCTGCA aeruginosa Pseudomonas ACTAGAAACTTTCCAGAGATGGATTGGTG--CCTTCGGGAATTTAGATACAGGTGCTGCA calcoaceticus Acinetobacter *********** * * *
Figure 1 (continued)
TGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT aureus Staphylococcus epidermidis Staphylococcus TGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT TGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCO pneumoniae Streptococcus WO
agalactiae Streptococcus TGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCC TGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCC pyogenes Streptococcus TGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCO faecalis Enterococcus faecium Enterococcus TGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCC TGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT mirabilis Proteus TGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT coli Escherichia marcescens Serratia TGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCI aerogenes Enterobacter TGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT TGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCC cloacae Enterobacter pneumoniae Klebsiella TGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT aeruginosa Pseudomonas TGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCC' TGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT calcoaceticus Acinetobacter **** aureus Staphylococcus TAAGCTTAGTTGCCATCATT--AAGTTGGGCACTCTAAGTTGACTGCCGGTGACAAAC epidermidis Staphylococcus TAAGCTTAGTTGCCATCATT--AAGTTGGGCACTCTAAGTTGACTGCCGGTGACAAACC TATTGTTAGTTGCCATCATT--CAGTTGGGCACTCTAGCGAGACTGCCGGTAATAAACCG pneumoniae Streptococcus TATTGTTAGTTGCCATCATT--AAGTTGGGCACTCTAGCGAGACTGCCGGTAATAAACCG agalactiae Streptococcus TATTGTTAGTTGCCATCATT--AAGTTGGGCACTCTAGCGAGACTGCCGGTAATAAACCO pyogenes Streptococcus TATTGTTAGTTGCCATCATT--TAGTTGGGCACTCTAGCGAGACTGCCGGTGACAAACCG faecalis Enterococcus ATTGTTAGTTGCCATCATT--CAGTTGGGCACTCTAGCAAGACTGCCGGTGACAAACCO faecium Enterococcus TATCCTTTGTTGCCAGCACGTAATGGTGGGAACTCAAAGGAGACTGCCGGTGATAAACCG mirabilis Proteus PATCTTTTGTTGCCAGCGGT-CCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTG coli Escherichia TATCCTTTGTTGCCAGCGGT-TCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTG marcescens Serratia TATCCTTTGTTGCCAGCGGT-CCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTG aerogenes Enterobacter TATCCTTTGTTGCCAGCGGT-CCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTO cloacae Enterobacter pneumoniae Klebsiella TATCCTTTGTTGCCAGCGGT-TAGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTG aeruginosa Pseudomonas GTCCTTAGTTACCAGCACC-TCGGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCG TTCCTTACTTGCCAGCATT-TCGGATGGGAACTTTAAGGATACTGCCAGTGACAAACTG calcoaceticus Acinetobacter ******
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(continued) 1 Figure aureus Staphylococcus GAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGO epidermidis Staphylococcus GAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTG GAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTG0 pneumoniae Streptococcus WO agalactiae Streptococcus GAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGC GAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGO pyogenes Streptococcus faecalis Enterococcus GAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGO faecium Enterococcus PAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTO mirabilis Proteus GAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGT0 coli Escherichia GAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGO marcescens Serratia GAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGC GAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTG aerogenes Enterobacter cloacae Enterobacter GAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTG pneumoniae Klebsiella GAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGC aeruginosa Pseudomonas GAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGO calcoaceticus Acinetobacter AGGAAGGCGGGGACGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGO ******
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******** * aureus Staphylococcus TACAATGGACAATACAAAGGGCAGCGAAACCGTGAGGTCAAGCAAATCCCATAAAGTTGT epidermidis Staphylococcus TACAATGGACAATACAAAGGGCAGCGAAACCGCGAGGTCAAGCAAATCCCATAAAGTTGT TACAATGGCTGGTACAACGAGTCGCAAGCCGGTGACGGCAAGCTAATCTCTTAAAGCCA0 pneumoniae Streptococcus agalactiae Streptococcus TACAATGGTTGGTACAACGAGTCGCAAGCCGGTGACGGCAAGCTAATCTCTTAAAGCCAA TACAATGGTTGGTACAACGAGTCGCAAGCCGGTGACGGCAAGCTAATCTCTTAAAGCCAA pyogenes Streptococcus faecalis Enterococcus TACAATGGGAAGTACAACGAGTCGCTAGACCGCGAGGTCATGCAAATCTCTTAAAGCTTO faecium Enterococcus ACAATGGGAAGTACAACGAGTTGCGAAGTCGCGAGGCTAAGCTAATCTCTTAAAGCTTO mirabilis Proteus CACAATGGCAGATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGAACTCATAAAGTCT coli Escherichia ACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCC TACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTATO marcescens Serratia TACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTATO aerogenes Enterobacter cloacae Enterobacter TACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCG pneumoniae Klebsiella TACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTATG aeruginosa Pseudomonas ACAATGGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGA TACAATGGTCGGTACAAAGGGTTGCTACCTAGCGATAGGATGCTAATCTCAAAAAGCCGA calcoaceticus Acinetobacter *
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(continued) 1 Figure aureus Staphylococcus TCTCAGTTCGGATTGTAGTCTGCAACTCGACTACATGAAGCTGGAATCGCTAGTAATCG epidermidis Staphylococcus TCTCAGTTCGGATTGTAGTCTGCAACTCGACTATATGAAGCTGGAATCGCTAGTAATCG TCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCG pneumoniae Streptococcus agalactiae Streptococcus TCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCG TCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCG pyogenes Streptococcus TCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCG faecalis Enterococcus TCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCG faecium Enterococcus WODiscount
TCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGT mirabilis Proteus TCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCG coli Escherichia TCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGT marcescens Serratia TCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGT aerogenes Enterobacter TCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGT cloacae Enterobacter pneumoniae Klebsiella TCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCG aeruginosa Pseudomonas TCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGT TCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGC calcoaceticus Acinetobacter *****
** AGATCAGCATGCTACGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCA0 aureus Staphylococcus epidermidis Staphylococcus AGATCAGCATGCTACGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCA GGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCAC pneumoniae Streptococcus Schoolings GGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCAC agalactiae Streptococcus GGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCA pyogenes Streptococcus GGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCA faecalis Enterococcus GGATCAGCACGCCGC-GTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCAO faecium Enterococcus AGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCA mirabilis Proteus coli Escherichia GGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCAT AGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCA marcescens Serratia AGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCAT aerogenes Enterobacter AGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACC cloacae Enterobacter pneumoniae Klebsiella AGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACC aeruginosa Pseudomonas GAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACC GGATCAGAATGCCC--GGTGATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCAT calcoaceticus Acinetobacter * MEMBERSHIP
Figure 1 (continued) aureus Staphylococcus GAGAGTTTGTAACACCCGAAGCCGGTGGAGTAACCTTTTAGGAGCTAGCCGTCGAAGGTG epidermidis Staphylococcus GAGAGTTTGTAACACCCGAAGCCGGTGGAGTAACCATT-TGGAGCTAGCCGTCGAAGGTG GAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCG-TAAGGAGCCAGCCGCCTAAGGTG pneumoniae Streptococcus WO agalactiae Streptococcus GAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCTAAGGTO GAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTATTAGGAGCCAGCCGCCTAAGGTC pyogenes Streptococcus faecalis Enterococcus GAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTTGGAGCCAGCCGCCTAAGGTG faecium Enterococcus GAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTT-TGGAGCCAGCCGCCTAAGGT mirabilis Proteus GGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTC-GGGAGGGCGCTTACCACTT coli Escherichia GGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTC-GGGAGGGCGCTTACCACTTTG marcescens Serratia GGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTC-GGGAGGGCGCTTACCACTTTG aerogenes Enterobacter GGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTC-GGGAGGNCGCTTTACCACT7 cloacae Enterobacter GGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTC-GGGAGGGCGCTTACCACTTTG pneumoniae Klebsiella GGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTC-GGGAGGGCGCTTACCACTTTG aeruginosa Pseudomonas GGAGTGGGTTGCTCCAGAAGTAGCTAGTCTAACCGCA-AGGGGGACGGTTACCACGGA calcoaceticus Acinetobacter GGGAGTTTGTTGCACCAGAAGTAGGTAGTCTAACCGCA-AGGAGGACGCTTACCACGGTG *****
* *
* *
**** aureus Staphylococcus GGACAAATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATO epidermidis Staphylococcus GGACAAATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATO GGATAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAG pneumoniae Streptococcus Shortness agalactiae Streptococcus GGATAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGG pyogenes Streptococcus GGATAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATC faecalis Enterococcus GGATAGATGATTGGGGTGAAGTCGTAACAAGGTAGCC--- faecium Enterococcus GGATAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCTGAAGGTGCGGCTGGATC mirabilis Proteus GATTCATGACTGGGGTGAAGTCGTAACAAGGTAACCGTAGGGGAACCTGCGGTTGGATO coli Escherichia TGATTCATGACTGGGGTGAAGTCGTAACAAGGTAACCGTAGGGGAACCTGCGGTT marcescens Serratia GATTCATGACTGGGGTGAAGTCGTAACAAGGTAACCGTAGGGGAACCTGCGG aerogenes Enterobacter cloacae Enterobacter TGATTCATGACTGGGGTGAAGTCGTAACAAGGTAACCGTAGGGGAACCTGCGGCTGGAT0 pneumoniae Klebsiella PGATTCATGACTGGGGTGAAGTCGTAACAAGGTAACCGTAGGGGAACCTGCGGTTGGATO TGATTCATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGGGAACCTGCGGCTGGATC aeruginosa Pseudomonas calcoaceticus Acinetobacter TGGCCGATGACTGGGGTGAAGTCGTAACAAGGTAACCA (continued) 1 Figure
WO 1426
Figure 1 (continued)
ACCTCCTTTCTACCTCCTTTCT
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